book of abstracts. albany 2019: the 20th conversationabstracts · lography. advances in single...

Book of Abstracts. Albany 2019: The 20th Conversation

Abstracts

1. The structure and dynamics ofbiomolecules in their native states

Joachim FrankDepartment of Biochemistry and Molecular Biophysics, ColumbiaUniversity, New York, NY 10032, USA

[email protected]

The aim of structural biology is to explain life processes interms of macromolecular interactions in the cell. These inter-actions typically involve more than two partners, and canrun up to dozens. A full description will need to characterizeall structures on the atomic level, and the way these struc-tures change in the process. Because of the crowded envir-onment of the cell, such characterization is presently (butsee below) only possible when the group of interacting mol-ecules (often organized into processive ‘molecular machines’)is isolated and studied in vitro. While X-ray crystallographyhas provided structures of a large number of molecularstructures, the need for crystals diffracting to high resolutionhas severely limited the number of supramolecular assem-blies and the range of conformers that can be studied withthis technique. Single-particle cryo-electron microscopy isabout to fill this gap, allowing functional processes to bestudied in great detail without imposing restraints on thestructures. There are many examples now for this expansionof Structural Biology toward a full characterization of a func-tional process. Future developments of single-particle cryo-EM include the study of short-lived intermediates in a none-quilibrium system by time-resolved techniques, and the char-acterization of continuous structural changes using datamining from large ensembles of molecule images. It is veryinteresting and promising that another technique of cryo-EM,cryo-electron tomography of FIB-milled cell sections, hasstarted to contribute information about processes evenwithin the cellular context. Distinct ribosome states, forinstance, have already been identified and localized by sub-tomogram averaging. With this advance, we get closer to thefulfillment of the most ambitious aim of Structural Biology,the visualization and interpretation of molecular interactionsin situ.

2. Cryo-EM and drug discovery

Sriram SubramaniamDjavad Mowafaghian Centre for Brain Health, 2215 Wesbrook Mall,University of British Columbia, Vancouver, BC, V6T 1Z3, Canada

[email protected]

Cryo-EM has transitioned rapidly in the last few years from beinga method that was only capable of handling a small subset ofcryo-EM worthy specimens, to one that is useful for analysis of avery large spectrum of protein complexes (Subramaniam, 2019).This transition of cryo-EM from being a technology that was billedas a tool to analyze large and/or highly symmetric specimens, toone that can successfully tackle a range of proteins and proteincomplexes of broad general interest has been transformative. Thestructures of an impressive number of proteins, small and large,sometimes with extensive conformational spread, have been suc-cessfully analyzed by cryo-EM. Many of these protein complexesmay never be coaxed to produce well-ordered crystals for studyby X-ray crystallography. It is important to recognize that inalmost every instance, these selected successes in the applicationof cryo-EM rest on decades of advances in biochemistry, biophys-ics and protein science that laid the necessary groundwork. Norcan we overlook the fact that the landscape of macromolecularentities that are still intractable to analysis by cryo-EM remainsimmense. Yet, the future looks bright, and there is every reasonto hope that an increasingly complex array of biological assem-blies will be tackled by cryo-EM.

Work in my laboratory has focused on the application ofcryo-EM to small dynamic protein assemblies, with particularemphasis on its use for drug discovery and therapeutic applica-tions, and for the development of image processing methods toimprove resolution (Banerjee et al., 2016; Chittori et al., 2018;Guo et al., 2017; Kang et al., 2018; Meyerson et al., 2016). In mypresentation, I will discuss recent examples where we have usedcryo-EM in this context to study metabolic enzyme complexes,ion channels, nucleic-acid protein complexes and intact viruses.

References

Banerjee, S., Bartesaghi, A., Merk, A., Rao, P., Bulfer, S. L., Yan, Y., Green,N., … Subramaniam, S. (2016). 2.3 Å resolution cryo-EM structure of

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS2019, VOL. 37, NO. S1, 1–92https://doi.org/10.1080/07391102.2019.1604468

human p97 and mechanism of allosteric inhibition. Science, 351(6275),871–875.

Chittori, S., Hong, J., Saunders, H., Feng, H., Ghirlando, R., Kelly, A. E., …Subramaniam, S. (2018). Structural mechanisms of centromericchromosome recognition by the kinetochore protein CENP-N. Science,359(6373), 339–343.

Guo, T., Bartesaghi, A., Yang, H., Falconieri, V., Rao, P., Merk, A., …Subramaniam, S. (2017). Cryo-EM structures reveal mechanism andinhibition of DNA targeting by a CRISPR-Cas surveillance complex.Cell, 171(2), 414–426.

Kang, Y., Kuybeda, O., de Waal, P. W., Mukherjee, S., Van Eps, N., Dutka,P., … Xu, H. E. (2018). Cryo-EM structure of human rhodopsin boundto an inhibitory G protein. Nature, 558(7711), 553–558.

Meyerson, J. R., Chittori, S., Merk, A., Rao, P., Han, T. H., Serpe, M., …Subramaniam, S. (2016). Structural basis of kainate subtype glutamatereceptor desensitization. Nature, 537(7621), 567–571.

Subramaniam, S. (2019). The cryo-EM revolution: Fueling the next phase.IUCrJ, 6(Pt 1), 1–2.

3. Deadly spiders and scary zombies—Not a Halloween story a near-atomicresolution glance into the CNS

Moran Shalev-BenamiDepartment of Structural Biology, Weizmann Institute of Science,Rehovot, 7610001, Israel [email protected]

Synapses are specialized junctions between neurons that trans-mit and compute information in the central nervous system(CNS). The establishment, properties, and dynamics of synapsesare governed by diverse trans-synaptic signaling molecules thatcommunicate their signal via multifarious interactions with theirsynaptic partners. Mutations in the genes encoding these mole-cules have been associated with diverse neuropsychiatric andneurodegenerative disorders thus highlighting their crucialimportance for normal brain function. Over the past few deca-des, tremendous efforts have been made to structurally char-acterize the trans-synaptic signaling molecules as well as theirinteracting partners. Nevertheless, their low expression levelsand high structural complexity has posed a great challenge totraditional structural methods, such as NMR and X-ray crystal-lography. Advances in single particle electron cryo-microscopy(cryo-EM) now allow the capture of such complexed macro-molecular assemblies in great details, providing snapshots ofthese fascinating molecules in action. Here we present thenear-atomic resolution structures of two such synaptic compo-nents, the cannabinoid receptor 1 (CB1R), and teneurin, twotransmembrane receptors that are primarily expressed in neu-rons and are considered to mediate various functions in syn-apse formation and maintenance. The structures provide ahigh-resolution glance into the receptors’ architectures andpresent structural insights into the interaction with their inter-and intra-cellular partners. Our results highlight cryo-EM as ahighly effective alternative approach for studying challengingmacromolecular machineries while providing a framework forelucidating the mechanisms of action of trans-synaptic signal-ing molecules that could in turn be used for design of futurenovel therapeutics.

4. Distance sensitive D-loop dynamicsand near-atomic resolution of F-actinphalloidin structure by cryoEM

Sanchaita Dasa!, Peng Gec,d!, Zeynep A. OztugDurera!, Elena E. Grintsevicha, Z. Hong Zhouc,d andEmil Reislera,baDepartment of Chemistry and Biochemistry, UCLA, Los Angeles,CA, USA; bMolecular Biology Institute, UCLA, Los Angeles, CA,USA; cDepartment of Microbiology, Immunology and MolecularGenetics, University of California, Los Angeles (UCLA), Los Angeles,CA, USA; dCalifornia NanoSystems Institute (CNSI), UCLA, LosAngeles, CA, USA [email protected]!Contributed equally.

Actin is indispensable for eukaryotic cells. Therefore, moleculardetails of F-actin structure and dynamics are essential for ourunderstanding of its key cellular functions. It is well establishedthat nucleotide-bound state of F-actin (ATP/ADP-Pi or ADP)defines its dynamic properties, stability and interactions withregulatory factors. Previous studies also indicate that aninnately flexible DNase I binding loop (D-loop, residues 40–50)plays a major role in such conformational dynamics. Recentadvances in cryoEM provide high-resolution information aboutnucleotide bound states of actin, however, the role of D loop inmonomer to polymer transition still remains elusive.Intriguingly, phalloidin, a ‘gold standard’ for actin staining invivo and in vitro, is also known to stabilize actin filaments andaffect the D-loop. Specifically, it can also convert polymeriza-tion-defective D loop mutants into stable polymers, therebybypassing D loop dependency. By utilizing a multidisciplinaryapproach of mutational disulfide crosslinking, light scatteringmeasurements and cryoEM, we probe the structural mecha-nisms that govern D-loop transitions in actin dynamics andhow phalloidin stabilizes F-actin. Our biochemical data providesa molecular ruler-based model of how intraprotomer distancebetween two D-loop residues facilitates a transition from G toF-actin and vice versa. Additionally, we report the first 3.7Åresolution structure of Phalloidin bound F-actin in the ADP-Pistate. Our structure supports (i) the role of methylation of His73 on actin in Pi binding, (ii) shows that phalloidin inhibits Pi

Figure 1. CryoEM structure of ADP-BeFx-F-actin-phalloidin. (a) Overall structureof ADP-BeFx-F-actin-phalloidin at 3.7 Å resolution, fitted with its atomic model.Red characters mark the relative position of actin subunits in the filament. (b)Representative regions (marked by amino acid numbers) of the density map fit-ted with their atomic models, showing the quality of the map (Figure 2).

2 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION

release from actin filaments via their direct interaction and (iii)defines phalloidin binding site on F-actin filaments and revealshow it restricts the relative movement between the two protofi-laments. Together, our results provide new molecular details ofF-actin structure and D loop dynamics. This structural and func-tional information may be also useful for designing phalloidinderivatives and selecting best phalloidin matching conjugatefor staining different actin forms and structures (Figure 1).

Sanchaita obtained her PhD in 2011 from Wesleyan Universityunder Dr. Donald Oliver. Then she did post-doctoral researchwith Dr. Joachim Frank, Columbia, Steven Doxsey and DavidLambright, Univ of Mass. Currently she is an assistant projectscientist with Dr. Emil Reisler at UCLA.

5. MicroED: conception, practice andfuture opportunities

Tamir GonenHoward Hughes Medical Institute and University of California, LosAngeles, Los Angeles, CA 90095, USA [email protected]

My laboratory studies the structures of membrane proteins thatare important in maintaining homeostasis in the brain.Understanding structure (and hence function) requires scientiststo build an atomic resolution map of every atom in the protein ofinterest, that is, an atomic structural model of the protein of inter-est captured in various functional states. In 2013, we unveiled themethod Microcrystal Electron Diffraction (MicroED) and demon-strated that it is feasible to determine high-resolution proteinstructures by electron crystallography of three-dimensional crys-tals in an electron cryo-microscope (CryoEM) (Nannenga, 2014;Shi, 2013). The CryoEM is used in diffraction mode for structuralanalysis of proteins of interest using vanishingly small crystals.The crystals are often a billion times smaller in volume than whatis normally used for other structural biology methods like X-raycrystallography. In this seminar, I will describe the basics of thismethod, from concept to data collection, analysis and structuredetermination, and illustrate how samples that were previouslyunattainable can now be studied by MicroED. I will conclude byhighlighting how this new method is helping us understandmajor brain diseases like Parkinson’s disease (Rodriguez, 2015);helping us discover and design new drugs; shedding new lighton chemical synthesis and small molecule chemistry; and show-ing us unprecedented level of details with subatomic resolutions.

Figure 2. Phalloidin can rescue the polymerization of oxidized double D-loop mutant F-C41C45. (a) Yeast actin mutant C41C45, with two cyteines in the D-loop, waspolymerized in the presence of 2mM MgCl2 as monitored by light scattering. Upon polymerization 20lM CuSO4 was added to catalyze the disulfide bond formationbetween C41 and C45. Finally, the polymerization of destructed filaments was rescued by the addition of equimolar phalloidin. Arrows indicate the described addi-tions to the actin sample. (b) Electron micrographs of F-C41C45 before and after oxidation. Electron microscopy samples were taken from experiments ran in parallelto the one shown in A. For control filaments, 10mM TCEP was added to ensure disulfide bond reduction. The scale bar in control image represents 0.2 mm.

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Funding

This research has been supported by funds from the HowardHughes Medical Institute.

References

Nannenga, B., Shi, D., Leslie, A. G. W., & Gonen, T. (2014). Continuousrotation structure determination by MicroED. Nature Methods, 11 (9),927–930.

Rodriguez, A. J., Ivanova, M., Sawaya, M. R., Cascio, D., Reyes, F., Shi, D.,… Eisenberg, D. (2015). The toxic core of a-synuclein of Parkinson’sdisease: Structure from invisible crystals. Nature, 525 (7570), 486–490.

Shi, D., Nannenga, B., Iadanza, M. G., & Gonen, T. (2013). MicroED—Threedimensional electron crystallography of protein microcrystals. eLife2,e01345: 1–e01317.

6. RNA helicase activity of E. coli HflXis instrumental in rescuing heat-inactivated 23S ribosomal RNA

Sandip Dey, Krishnamoorthi Srinivasan andJayati SenguptaStructural Biology & Bio-Informatics Division, CSIR—IndianInstitute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata,700 032, India [email protected]

A recent study has revealed that the ribosome-associatedGTPase HflX acts as an anti-association factor upon binding

to the 50S subunit during heat stress. Although HflX is rec-ognized as a GTPase, several studies have shown that E. coliHflX is capable of hydrolyzing ATP as well and its N-terminaldomain 1 has recently been characterized as the ATPasedomain. However, the functional role of its ATPase activityremains unknown.

Here, using biochemical assays and atomic force micros-copy, we demonstrate for the first time, that E. coli HflX pos-sesses ATP-dependent RNA helicase activity and is capable ofunwinding large subunit ribosomal RNA. A cryo-EM structureof the 50S-HflX complex in the presence of ATP and GTP(non-hydrolysable analogs) hinted at the mode of its actionas an RNA helicase, where a helical domain has a determin-ant role in RNA unwinding. We further show that, whileheat-stress results in inactivation of the ribosome, HflX canrestore heat-damaged ribosomes and, consequently, amelior-ate cell survivability.

References

Coatham, M. L., Brandon, H. E., Fischer, J. J., Sch€ummer, T., & Wieden,H. J. (2016). The conserved GTPase HflX is a ribosome splitting factorthat binds to the E-site of the bacterial ribosome. Nucleic AcidsResearch, 44(4), 1952–1961.

Dey, S., Biswas, C., & Sengupta, J. (2018). The universally conservedGTPase HflX is an RNA helicase that restores heat-damagedEscherichia coli ribosomes. The Journal of Cell Biology, 217(7),2519–2529.

Zhang, Y., Mandava, C. S., Cao, W., Li, X., Zhang, D., Li, N., … Gao, N.(2015). HflX is a ribosome-splitting factor rescuing stalled ribosomesunder stress conditions. Nature Structural & Molecular Biology, 22(11),906–913.


7. Supercoiling affects specific baseaccessibility to alter 3-D shape ofDNA minicircles

Jonathan M. Fogg and Lynn ZechiedrichDepartment of Molecular Virology and Microbiology, BaylorCollege of Medicine, Houston, TX 77030, USA [email protected]

DNA supercoiling affects DNA metabolism yet much abouthow it does so is unknown (reviewed in Fogg et al., 2012).Using 336 bp DNA minicircles covering a range of positive tonegative supercoiling, we unveiled the first three-dimensionalstructures of supercoiled DNA using cryo-electron tomography(Irobalieva et al., 2015). With supercoiling, DNA can form farmore bent and contorted shapes than predicted. We sought tounderstand how the interplay of DNA sequence and supercoil-ing drives the formation of these shapes using coarse-grainedsimulations and biochemical probing. Base pair disruptionsindicate regions of high bending, as localized denaturation(molecule dynamic simulations indicate from base flipping;Randall et al., 2009) creates flexible hinges. At the same time,sharp bending at the apices of highly writhed DNA circles leadsto broken base pairs. Probing with nuclease Bal-31 revealedexposed bases as a function of supercoiling. Bal31 cleaved allthe negatively supercoiled 336 bp minicircles but the rateincreased beyond a distinct negative supercoiling threshold.This threshold shifted to more negative supercoiling for 672 bpminicircles with inherently less curvature, demonstrating therelationship between bending and base accessibility. A sharppositive supercoiling threshold was required for Bal-31 cleavageto occur. We mapped Bal-31 cleavage sites and, using coarse-grained simulations, determined the DNA register of our cryo-electron micrograph images. Our data reveal three hotspots ofBal-31 cleavage; two are located " 180# apart along the DNA cir-cumference, and another is seen only in minicircles with lownegative supercoiling levels. The relative probability of Bal-31cleaving at either site varied as a function of supercoiling.Together these data reveal the interplay among sequence, super-coiling and shape, resulting in conformational changes thatshould profoundly influence DNA interactions with proteins.

Understanding these changes could facilitate the design ofsupercoiling-dependent DNA nanostructures for gene therapy.

Funding

This work was supported by NIH grant RO1GM115501.

References

Fogg, J. M., Randall, G. L., Sumners, D. W. L., Pettitt, B. M., Harris, S. A., &Zechiedrich, L. (2012). Bullied no more: When and how DNA shovesproteins around. Quarterly Reviews of Biophysics, 45(03), 257–299.

Irobalieva, R. N.,! Fogg, J. M.,! Catanese, D. J., Sutthibutpong, T., Chen, M.,Barker, A. K., … Zechiedrich, L. (2015). Structural diversity of super-coiled DNA. Nature Communications, 12 6, 8440 (!co-first authors)

Randall, G. L., Zechiedrich, L., & Pettitt, B. M. (2009). In the absence ofwrithe, DNA relieves torsional stress with localized, sequence-depend-ent structural failure to preserve B-form. Nucleic Acids Research,37(16), 5568–5577.

8. Understanding the functionaldynamics of the 26S proteasome bynucleotides-proteasomeinteraction study

Rui Fanga and Ying LubaDepartment of Molecular and Cellular Biology, Harvard University,Cambridge, MA 02138, USA; bDepartment of Systems Biology,Harvard Medical School, Boston, MA 02115, USA

[email protected]

The 26S proteasome, consisting of a barrel-shaped proteo-lytic 20S core complex and one or two 19S regulatory com-plexes, is a macromolecular machine (2.5 MDa) responsiblefor regulatory protein degradation in eukaryotic cells. Theproteasome has a hexameric ring of AAAþATPases at thebottom of the 19S proteasome which converts ATP’s chem-ical energy to mechanical force to unfold protein substratesand translocate the denatured polypeptide through the cen-tral pore into the 20S for degradation. Though genetic, bio-chemical and structural studies have revealed great detailsabout the proteasome, how the six ATPases in the 19S par-ticle coordinate their ATP cycles to power substrate trans-location and conformational transitions of other parts of theproteasome remains elusive. To understand this question, wedeveloped a fluorescent reporter with sub-nanomolar sensi-tivity to measure different aspects of proteasomal activities.In a competition assay, we found that ATP-cS inhibited sub-strate degradation much more strongly compared to ADP,and primarily affected the rate of substrate translocation. Wethen developed a single-molecule assay to study the kineticsof the nucleotides-proteasome interaction. By labelingnucleotides with fluorophores, we are able to detect singlenucleotide binding and disassociation events under a TIRFmicroscope. The result shows that proteasomal ATPases arecharacterized by distinct nucleotide binding kinetics.Interestingly, the overall binding kinetics of ATP is similar tothat of ADP and ATP-cS, while AMP binds rather weakly. Toexplain the different inhibitory effects of nucleotides in thecompetition assay, we devised a Markov-state model


representing all possible transition of the proteasomal ATPases.Simulation of this model suggests an entropy-driven mechanismunderlying the inhibition by nucleotide inhibitors, which may beadaptive under low ATP conditions. Our study provides novelunderstanding of the working principles of proteasomalATPases, with the goal to obtain mechanistic insights by bridgingthe recent structural advances and biochemical observations.

9. Structural and quantitative studyof the destruction complex in thecanonical wnt-signaling pathway

Bai Luan, Wenzhe Ma, Hong Kang, Ying Lu andMarc W. KirschnerDepartment of Systems Biology, Harvard Medical School, 200Longwood Ave, Boston, MA 02115, USA

[email protected]

The canonical Wnt/b-catenin signaling pathway plays pivotalroles in tumorigenesis. The cytosolic level of b-catenin ismainly regulated by a postulated molecular machine, which iscalled the b-catenin destruction complex. However, while theterm molecular machine usually connotes a discrete multipro-tein complex like the ribosome, this machine, while stronglysupported indirectly has never been purified or seen. Thedestruction complex may be transient, and its postulated stoi-chiometry may never be realized at any given instant. Theinstability of the components may lead to many different kin-etically determined structures. The variable stoichiometrycould also be very relevant physiologically. This complex con-tains a large scaffold, the adenomatous polyposis coli protein(APC), which acts as an important tumor suppressor and hasbeen well characterized in colorectal cancer. APC mutants innearly all colorectal cancer have variable truncations, whichcould produce different subunit configurations between APCand b-catenin, influencing the degradation of b-catenin down-stream. To study the dynamic stoichiometry, we are develop-ing a quantitative single-molecule assay in the cell extracts ofboth normal cells and colorectal cancer cells to observe theassembly process with millisecond time resolution. Ultimately,combining this kinetic information with biochemical crosslink-ing and cryo-electron microscopy, we hope to capture thetransient state of the destruction complex of kinetically stablesubcomplexes. The purpose of the project is to illustrate thedynamic process of the destruction complex by combiningstructural information and quantitative single-molecule data,aiming to find the essential role of APC in colorectal cancer.

10. The conformational landscape ofthe human 26S proteasome by Cryo-EM structural analysis

Rui Fanga, Robin Chenb, Jason Hona, Jiayi Wub,Yuanchen Dongb, Yanan Zhub, Youdong Maob, QiOuyangb, Daniel Finleyc and Ying Lua

aDepartment of Systems Biology, Harvard Medical School, Boston,MA 02115, USA; bState Key Laboratory for Artificial Microstructuresand Mesoscopic Physics, School of Physics, Peking University,Beijing, China; cDepartment of Cell Biology, Harvard MedicalSchool, Boston, MA 02115, USA [email protected]

The 26S proteasome is a macromolecular machine (2.5-MDa)that mediates ubiquitin-dependent protein degradation in alleukaryotic species. The proteasome consists of a barrel-shaped20S core complex flanked by two hexameric rings ofAAAþATPases that harvest the energy from ATP hydrolysis tounfold and translocate substrates through the central poreinto the degradation chamber. In a previous study, we deter-mined how the ubiquitin configurations on substrates dictatedthe degradation rate by the proteasome. Currently, we aim tounderstand how the 26S proteasome recognizes these ubiqui-tin configurations and commits the substrate to a processivedegradation process. In a series of studies, we used moderncryo-EM technology and classification algorithms to identifymultiple conformations of the proteasomes, which provideimportant insights into these questions. To complement thelack of kinetic information in structural analysis, we developeda fluorescent reporter with sub-nanomolar sensitivity tomeasure proteasomal activities under different conditions,and established single-molecule assays to detect the transi-ent interactions between nucleotides and the proteasomeusing a TIRF microscope. To synergize these results, we cre-ated a rule-based model, inspired by comparative analysis ofthe proteasome structures, which simulates the dynamictransitions of a translocating proteasome on a complete con-formational landscape with unknown parameters that can beobtained from kinetic studies. Suggested by the results, apathway of sequential loading/hydrolysis emerges with thehighest probability, and minor pathways with lower proba-bilities also exist which may become the major one and con-sequently lower the degradation rates as in differentproteasomal mutants. In summary, our study promotes adeeper understanding of the functional dynamics of the pro-teasome driven by ATP hydrolysis, with the goal to obtainmechanistic insights by bridging the recent structural advan-ces and biochemical observations.

11. Computational design of DNAscaffold for optimization of CryoEM

Kendar Serindaga,b, Kelly M. Thayerb,c,d, David LBeveridgeb,c and David R. Langleyb,c,eaDepartment of Molecular Biology and Biochemistry, WesleyanUniversity, Middletown, CT 06459, USA; bDepartment ofChemistry, Wesleyan University, Middletown, CT 06459, USA;cMolecular Biophysics Program, Wesleyan University, Middletown,CT 06459, USA; dDepartment of Computer Science, WesleyanUniversity, Middletown, CT 06459, USA; eArvinas, New Haven, CT06511, USA

The ability of DNA to bind proteins and other moleculesmakes it an ideal candidate for its use as a scaffold to which


biomolecules can be attached. In this study, circular DNAscaffolds of varying sizes have been designed. Curvature inthe DNA ring is induced by the bacterial DNA binding proteinIntegration Host Factor (IHF). It is a heterodimer with an alphahelical body and two protruding beta sheet arms. These armsinteract within the minor groove of DNA, and proline residueswithin the arms stabilize the DNA bending. Molecular dynamicsimulations were run to determine the mechanics and stabilityof the DNA ring itself, as well as the mechanics and stability ofIHF protein binding. Wet lab experiments are also being con-ducted to create the physical construct. Applications for thisscaffold include its use in cryo-electron microscopy (CryoEM), anew molecular imaging technique that enables researchers todetermine the structure of biomolecules. CryoEM requires bio-molecules to be at least 200 kDa and maintain an axis of sym-metry in order to obtain an accurate image of the structure.Thus, attaching biomolecules less than 200 kDa will enablethem to be readily visualized by CryoEM. Additional functional-ity of this scaffold includes the facilitation and subsequentimaging of weak binding interactions.

12. Effect of crown ether, 15-crown-5,on lysozyme amyloid fibrillation

Anirban BasuDepartment of Chemistry and Chemical Technology, VidyasagarUniversity, Midnapore, 721 102, India

[email protected]

Amyloid fibrils are highly organized protein or peptide aggre-gates with cross b-sheet rich secondary structure responsiblefor various pathological disorders such as type 2 diabetes,Alzheimer’s, Huntington’s and Parkinson’s diseases. Amyloid

fibrils can also be formed by several nonpathogenic proteinslike serum albumins and hen egg-white lysozyme under suit-able conditions in vitro. This suggests aggregation to be aninherent property of proteins. Although amyloid fibrillationcauses many fatal diseases, effective therapeutic treatmentsare still obscure. Various crown ethers have been shown toretard fibrillation effectively (Banik et al., 2016; Tian et al.,2014). Here, we have studied the effect of crown ether,namely 15-crown-5, on amyloid fibrillation in lysozyme inorder to design effective anti-amyloidogenic agents that cantarget native proteins and offer advantageous prospect todevelop therapeutics. Lysozyme is an antimicrobial enzymepresent in various tissues as well as protective secretionswhich can form amyloid fibrils readily and hence serves anexcellent model system for studying protein amyloidogenesis.In this work, hen egg-white lysozyme was taken as the modelprotein system since it shares " 60% sequence homology withhuman lysozyme that is linked with many hereditary non-neuropathic systemic amyloidosis. Various assays were under-taken to ascertain the inhibitory influence of the crown etheron lysozyme fibrillation. The results of Thioflavin T fluores-cence assay indicated that the crown ether can attenuate fib-rillation effectively. This observation was further reiterated bya complementary Congo Red Assay in absorbance. The kinet-ics of fibrillation was also studied using the Thioflavin T fluor-escence assay. Far-UV circular dichroism studies indicated thatthe lysozyme samples undergo a-to-b transition, upon amyl-oid fibrillation. From the far-UV circular dichroism studies itwas also inferred that the b-sheet content of the protein wasreduced in the presence of crown ether which suggested thatamyloid fibrillation was inhibited. Fluorescence microscopy,Nile red fluorescence assay, 8-anilino-1-naphthalenesulfonica-cid binding assay, intrinsic (tryptophan) fluorescence studiesalong with steady-state fluorescence anisotropy also testifiedthat the crown ether had significant fibril attenuating ability.Atomic force microscopy (AFM) imaging studies unequivocallyestablished that fibril formation was reduced in the presenceof the crown ether. Thus, the inhibitory effect of the crownether, 15-crown-5, on amyloidosis may be exploited fordesigning better therapeutics for the treatment of amyloido-genesis-related diseases.

Acknowledgments

Financial assistance from the Department of Science & Technology andBiotechnology, Govt. of West Bengal (File No.: ST/P/S&T/15G-13/2018) isgratefully acknowledged. The author is grateful to Prof. RanjanChakrabarti, Hon’ble Vice-Chancellor, Vidyasagar University, forhis patronage.

References

Banik, D., Dutta, R., Banerjee, P., Kundu, S., & Sarkar, N. (2016). Inhibitionof fibrillar assemblies of l-phenylalanine by crown ethers: A potentialapproach toward phenylketonuria. The Journal of Physical Chemistry B,120(31), 7662–7670.

Tian, Y., Zhang, X., Li, Y., Shoup, T. M., Teng, X., Elmaleh, D. R., … Ran, C.(2014). Crown ethers attenuate aggregation of amyloid beta ofAlzheimer’s disease. Chemical Communication (Cambridge, UK), 50(99),15792–15795.


13. Evaluation and validationof synergistic effect of predictedamyloid-beta (Ab) inhibitor by deepneural network

Aman Chandra Kaushik and Dong Quing WeiState Key Laboratory of Microbial Metabolism and School of lifeSciences and Biotechnology, Shanghai Jiao Tong University,Shanghai 200240, China [email protected]

Amyloid-beta 42 (Ab-42) protein has been established innumerous investigations as high-profile risk factor associatedwith onset and progression of Alzheimer’s disease (AD).Extracellular senile plaques accumulation, synaptic degener-ation and intracellular neurofibrillary tangles (NFT) wererecorded as essential features that facilitate onset of Ab-42and results into AD. Hence, this study attempted a newscreening technique to discover potential inhibitors againstAb-42 by employing in silico deep neural network approach.In this method, Pubchem compounds library was screenedand concluded wgx-50 as potential inhibitor of Ab-42. Also,synergistic effect of wgx-50þ gold nanoparticles (AuNP)induced significant inhibition of Ab-42 against wgx-50 ascontrol. Further, analysis of molecular docking, system biol-ogy approach and time course simulation revealed synergis-tic effect of wgx-50-AuNP complex as potential treatment forAD. Additionally, we purposed the biological circuit for theAD induced by Ab-42 that can be employed to monitor theeffect of drugs on AD (Figure).

Funding

This work is supported by the Key Research Area Grant2016YFA0501703 from the Ministry of Science andTechnology of China, State Key Lab on Microbial Metabolism,

and Joint Research Funds for Medical and Engineering fromScientific Research at Shanghai Jiao Tong University.

References

Austen, B. M., Frears, E. R., & Davies, H. (2000). The use of SeldiProteinChipTMArrays to monitor production of Alzheimer’s b-amyloidin transfected cells. Journal of Peptide Science, 6(9), 459–469.

Kaushik, A. C., Kumar, A., Dwivedi, V. D., Bharadwaj, S., Kumar, S., Bharti,K., … Mishra, S. K. (2018). Deciphering the biochemical pathway andpharmacokinetic study of amyloid beta-42 with superparamagneticiron oxide nanoparticles (SPIONs) using systems biology approach.Molecular Neurobiology, 55(4), 3224–3236.

14. How nature harnesses entropyto tune protein function

Zachary A. Wooda, Nicholas D. Keula, KrishnadevOrugantyb, Elizabeth T. Schaper Bergmane,Nathaniel R. Beattiea, Weston E. McDonalda, RenukaKadirvelraja, Michael L. Grosse, Robert S. Phillipsc andStephen C. HarveydaDepartment of Biochemistry and Molecular Biology, University ofGeorgia, Athens, GA, USA; bDepartment of BiomedicalEngineering, University of Michigan, Ann Arbor, MI, USA;cDepartment of Chemistry, Washington University in St. Louis, St.Louis, MO, USA; dDepartment of Chemistry, University of Georgia,Athens, GA, USA; eDepartment of Biochemistry and Biophysics,University of Pennsylvania, Philadelphia, PA, USA

[email protected]

How evolution shapes the conformational landscape of aprotein to tune a specific function is poorly understood.Protein evolution is constrained by the stability of the folded,native state. Despite this, many proteins contain intrinsicallydisordered (ID) peptide segments. In fact, 44% of human pro-teins contain ID segments >30 residues in length. The major-ity of these segments have no known function and are often

Figure.


removed to facilitate structural studies. Here, we show thatan ID segment can enhance the affinity of an effector bind-ing site by modifying the dynamics of an allosteric network.The enhanced affinity does not depend on the sequence orcharge of the ID segment. Instead, changes in effector bind-ing affinity can be accurately predicted based on segmentlength alone. Using a combination of transient state kinetics,hydrogen-deuterium exchange mass spectrometry, thermaldenaturation studies, computer simulation and crystal struc-ture analysis, we show that the ID segment alters the energylandscape of a folded protein to favor the allosteric response.Our evidence shows that the ID segment generates anentropic force that can rectify the conformational ensembleof a protein to favor a specific functional state. Thus, the per-sistence of intrinsic disorder in the proteome may reflect theevolution of low complexity structural elements that cantune a specific protein function.

References

Keul, N. D., Oruganty, K., Bergman, E. T. S., Beattie, N. R., McDonald,W. E., Kadirvelraj, R., … Wood, Z. A. (2018). The entropic force gener-ated by intrinsically disordered segments tunes protein function.Nature, 563(7732), 584.

15. Intrinsically disordered proteins,alternative splicing, and post-translational modification (IDP-AS-PTM): a toolkit fordevelopmental biology

Keith DunkerCenter for Computational Biology and Bioinformatics, Departmentof Biochemistry and Molecular Biology, Indiana University Schoolsof Medicine and Informatics, Indianapolis, IN, 46202, USA

[email protected]

Intrinsically, disordered proteins and regions (IDPs and IDRs)lack well-defined tertiary structures, yet carry out variousimportant cellular functions, especially those associated withcell signaling and regulation. In eukaryotes, IDPs and IDRscontain the preferred loci for both protein segmentsencoded by alternatively spliced pre-mRNA (AS) and manypost-translational modifications (PTMs). Furthermore, AS and/or PTMs at these loci generally alter the signaling outcomesassociated with these IDPs or IDRs. However, the prevalenceof such functional modulations remains unknown. Also, thesignal-altering mechanisms by which AS, and PTMs modu-late function and the extent to which AS and PTMs collab-orate in their signaling modulations have not been welldefined for particular protein examples. Here, we focus onthree important signaling and regulatory IDR-containingprotein families in humans, namely G-protein coupledreceptors (GPCRs), which are transmembrane signaling pro-teins, the nuclear factors of activated T-cells (NFATs), which

are transcription factors (TFs), and the Src family kinases(SFKs), which are signaling enzymes. The goal here is todetermine how AS and PTMs individually alter the out-comes of the signaling carried out by the various IDRs andto determine whether AS and PTMs work together to bringabout differential cellular responses. We also present dataindicating that a wide range of other signaling IDPs or sig-naling proteins containing IDRs also undergo both AS- andPTM-based modifications, suggesting that these many pro-teins likely take advantage of signal outcome modulationsthat result from collaboration among these three features.Hence, we propose that the widespread cooperation ofIDPs, AS and/or PTMs substantially contributes to, or evenprovides the basis for, the vast complexity of eukaryotic cellsignaling systems.

16. Multivalency regulates activityin an intrinsically disorderedtranscription factor

Sarah Clark, Kayla Jara, Patrick Reardon andElisar BarbarDepartment of Biochemistry and Biophysics, Oregon StateUniversity, Corvallis, OR 97331, USA [email protected]

Transcription factors contain multiple regulatory sites foreither post-translational modifications or binding partners,and their activity is thus tuned by the combined action ofthese components. Recent studies have revealed a highdegree of intrinsic disorder in transcription factors, indicatingthat the inherent dynamical behavior harbored by thesestructures is critical for these regulatory events to take place.Our developing understanding suggests that these intrinsic-ally disordered domains may provide a multivalent platformfor the recruitment of regulatory binding partners. Anexample of a transcription factor with a long disordereddomain is ASCIZ (ATMIN, ZNF822) which also has anunusually high number of multivalent sites for the productof its main target gene, the hub protein LC81. ASCIZ has afolded N-terminal zinc finger domain for DNA binding andan unusually long disordered C-terminal domain, which bindsmultiple copies of LC8: 11 in human and 7 in Drosophila pro-teins. Here, we integrate multiple approaches including NMRand single particle electron microscopy to elucidate thestructure, dynamics, thermodynamics and hydrodynamics ofthe large disordered ASCIZ-LC8 complexes, that togetherreveal a new model by which ASCIZ can maintain stablepools of the hub protein LC8. We tested the main features ofthis model in cells using transcription activity assays whichshow a trend wherein mutant ASCIZ constructs with lowerLC8 occupancy display higher transcriptional activity, whileconstructs with higher LC8 occupancy have lower activity.We propose that a dynamic ensemble of complexes isimportant for fine-tuning ASCIZ transcriptional activity, wherestable, low occupancy complexes function to maintain a basal


buffering transcription rate for LC8. A change in LC8 cellularconcentration would shift this dynamic equilibrium to ahigher or lower occupancy state without dramatically alter-ing the level of transcription. High LC8 occupancy shutsdown transcription while low LC8 occupancy turns on tran-scription2. As LC8 is an essential regulator of dozens of cellu-lar processes, an ability to maintain an LC8 ‘buffer’ is likelyimportant for cellular homeostasis. Although many othertranscription factors are regulated by multisite phosphoryl-ation or multiple binding events to different proteins wefind no examples of activity tuned by multivalent binding tothe gene product in a negative autoregulatory role, whichunderscores the novelty and potential impact of this study(Figure 1).

Funding

This research is supported by NSF MCB-1617019 and NIH GM084276 and HEI 1S10OD018518.

References

Barbar, E. (2008). Dynein light chain LC8 is a dimerization hub essentialin diverse protein networks. Biochemistry, 47(2), 503–508.

Clark, S. A., Myers, J. B., King, A., Fiala, R., Novacek, J., Pearce, G., …Barbar, E. (2018). Multivalency Regulates activity in an intrinsically dis-ordered transcription factor. eLife, 1, 7. pii: e36258.

17. Remembering the past: a newform of protein-based inheritance

Daniel F. JaroszDepartment of Chemical and Systems Biology, Stanford University,Stanford, CA, USA [email protected]

During their lifetimes, individuals commonly experience tran-sient changes in gene expression as a result of differentenvironmental stimuli. These responses are often thought tohave little heritable influence once they decay. However, wehave recently discovered that such stimuli frequently induce

self-perpetuating changes in protein conformations. Thisoccurs most commonly in intrinsically disordered proteinsthat regulate information flow: chromatin modifyingenzymes, transcription factors and RNA binding proteins.These conformations can be broadly defined as prions,although their structures do not usually match the cross-beta sheet amyloids of the archetypical prion PrP. However,like known prions, corresponding changes in protein func-tion are heritable from one generation to the next withoutany change to the genome. In this sense, such protein-basedinheritance represents an extreme form of epigenetics. Wehave begun to characterize the biochemistry of these ele-ments and investigate their influence on disease, develop-ment and evolution. Lessons learned provide insight intomechanisms of pathological and beneficial protein aggrega-tion alike, and how they might be modulatedtherapeutically.

18. Seeing the structure of RNA-binding proteins in membranelessorganelles and disease-associated aggregates

Nicolas Lux FawziMolecular Pharmacology, Physiology & Biotechnology, BrownUniversity, 70 Ship Street, Box G-E, Providence, RI, 02912-G, USA

[email protected]

Nicolas Fawzi, Brown University, leads a team using NMRspectroscopy, molecular simulation, microscopy and cellassays to probe disordered protein domain assembly andfunction. Disordered domains of RNA binding proteins aggre-gate in several neurodegenerative diseases and mediate for-mation of functional, liquid, membraneless organelles. Nick’sgroup probes the structure of these protein (including TDP-43, FUS, hnRNPA2) within in vitro models of these organelles,the mechanistic structural changes due to disease-causingmutations, the ability of post-translational modification toalter assembly.

19. The critical role of intrinsicdisorder in creatingbioactive materials

Sarah E. BondosDepartment of Molecular and Cellular Medicine, 440 ReynoldsMedical Building, Texas A&M Health Science Center, CollegeStation, TX 77843-1114, USA

The development of materials with diverse functional proper-ties enables a broad range of applications. For materialscomposed of protein, well-established molecular biologytechniques can theoretically be used to genetically fuse full-length functional proteins to the self-assembling protein, cre-ating a single amino acid chain capable of both forming

Figure 1. A collage of NMR spectra, electron micrograph and LC8/ASCIZ struc-ture showing three LC8 dimers and two chains of ASCIZ, while the rest of thecomplex remains disordered. Obtaining data of sufficient quality on a heteroge-neous system of multiple conformations of IDP complexes was made possibleby creative use of interdisciplinary approaches.


materials and enacting the function of interest. However,protein-based materials are typically assembled under dena-turing conditions, precluding incorporation of proteins in anactive state. Our lab discovered that the Drosophila mela-nogaster transcription factor Ultrabithorax (Ubx) self-assem-bles into nanoscale to macroscale materials. The disorderedregions of Ubx resemble the amino acid sequence of elastin,and the mechanical properties of Ubx fibers resemble thoseof elastin. Because Ubx materials rapidly form in mild, aque-ous buffers, a surprisingly wide variety of full-length mono-meric and multimeric proteins can be incorporated intoUbx materials via gene fusion without harming materialsassembly or impairing the function of the appended pro-tein. Indeed, fusing Ubx is fused to angiogenic growth fac-tors, creates fibers that control cell signaling and cellbehavior in vitro, and can instigate and guide blood vesselformation in vivo. Finally, Ubx fibers retain the ability tobind specific DNA sequences, and Ubx monomers bound toDNA can still assemble into fibers. The intrinsically disor-dered regions of Ubx allows incorporation of molecules upto three times the size of this protein. One-pot productionof functionalized Ubx materials provides a facile, scalableplatform for customizing materials for a variety ofapplications.

20. Complex self-assembly andtransformation of DNA nanostructures

Yonggang KeBiomedical Engineering Department, Emory University andGeorgia Institute of Technology, Atlanta, GA, 30322, USA

[email protected]

A key challenge in nanotechnology is to design and fabricatenanostructures and nanodevices, which can be used as gen-eral platforms for basic science research (e.g., material scien-ces, structural biology, molecular biology, etc.), and forpractical applications. Owing largely to its programmabledesign strategies, nucleic acids self-assembly, and in particu-lar DNA self-assembly, has emerged as a powerful approachin programming self-assembly of custom-designed intricatenanostructures.

The core mission of our lab (ke-lab.gatech.edu) is todevelop novel bottom-up self-assembly strategies to fullydemonstrate the potential of DNA as a programmable nano-material. Our most recent work focuses on making massive/complex static DNA nanostructures and dynamic DNA devi-ces. In 2012, we invented a modular assembly strategy forconstructing complex 3D shapes, up to 8 megadalton insize, using short synthetic DNA oligos—‘DNA bricks’. We willdiscuss how we can use this method to construct fullyaddressable, three-dimensional GDa nanostructures withrationally designed shapes. The second part of this talk willfocus on dynamic DNA nanomachines that can perform arange of controlled motions at nanoscale. Particularly, wehave demonstrated prescribed, long-range information relay

in artificial molecular arrays assembled from modular DNAantijunction units. The array transformation is equivalent to amolecular ‘Domino’: Once initiated at a few selected units,the transformation then propagates to neighboring unitsand eventually the entire array. The specific informationpathways by which this transformation occurs can be con-trolled by adding trigger strands to specific units, or by alter-ing the design of individual units, the connections betweenunits, and the geometry of the array. Beyond sophisticatednanostructures and nanomachines, DNA nanotechnology hasfound increasing capabilities in many applications, such asfabrication of functional materials at nanoscale precision. Asexamples of DNA-based applications, I will also present someof our most recent works on DNA-templated self-assembly ofnanoparticles, and on DNA-based nanoscale drug deliv-ery systems.

References

Ke, Y., Ong, L. L., Shih, W. M., & Yin, P. (2012). Three-dimensional struc-tures self-assembled from DNA bricks. Science (New York, N.Y.),338(6111), 1177–1183.

Ong, L. L., Hanikel, N., Yaghi, O. K., Grun, C., Strauss, M. T., Bron, P., Lai-Kee-Him, J., … , Yin, P. (2017). Programmable self-assembly of three-dimensional nanostructures from 10,000 unique components. Nature,552(7683), 72–77.

Song, J., Li, Z., Wang, P., Meyer, T., Mao, C., & Ke, Y. (2017).Reconfiguration of DNA molecular arrays driven by information relay.Science, 357(6349), eaan3377.

Wang, D., Song, J., Wang, P., Pan, V., Zhang, Y., Cui, D., & Ke, Y. (2018).Design and operation of reconfigurable two-dimensional DNAmolecular arrays. Nature Protocols, 5, 693.

21. Designer nucleic acid architecturesfor programmable self-assembly

Hao YanCenter for Molecular Design and Biomimetics, Biodesign Institute,Arizona State University, Tempe, AZ 85287, USA

[email protected]

DNA and RNA have emerged as an exceptional molecularbuilding block for nano-construction due to its predictableconformation and programmable intra- and inter-molecularbase pairing interactions. A variety of convenient designrules and reliable assembly methods have been developedto engineer DNA nanostructures of increasing complexity.The ability to create designer DNA architectures withaccurate spatial control has allowed researchers to explorenovel applications in many directions, such as directedmaterial assembly, structural biology, biocatalysis, DNAcomputing, nano-robotics, disease diagnosis and drugdelivery. In this talk, I will discuss some of our work inthe field of structural nucleic acid nanotechnology, andpresents some of the challenges and opportunities thatexist in DNA and RNA-based molecular design andprogramming.


22. Using DNA origami to decipherspatial effects in biology

Bj€orn H€ogbergDepartment of Medical Biochemistry and Biophysics, KarolinskaInstitute, Stockholm 171 77, Sweden [email protected]

It is widely accepted that the biophysical context of ligandsand receptors has significant impact of downstream signal-ing, however, the concept is poorly understood due to diffi-culties in controlling and analyzing the microenvironment onthe nanoscale. I will talk about how we think that DNA-nano-technology can be of great help in both learning the tactilealphabet of cells by stimulation using protein decoratedDNA-origami ‘nano-calipers’ and of help in understandingthe binding of antibodies. I will also briefly present ourrecent work on ‘3D-printing’ DNA origami wireframe struc-tures, a method that provides a way to make origami moreaccessible to experiments in physiological salt conditions.

References

Benson, E., Mohammed, A., Gardell, J., Masich, S., Czeizler, E., Orponen,P., & H€ogberg, B. (2015). DNA rendering of polyhedral meshes at thenanoscale. Nature, 523(7561), 441–444.

Shaw, A., Hoffecker, I. T., Smyrlaki, I., Rosa, J., Grevys, A., Bratlie, D., …H€ogberg, B. (2019). Binding to nanopatterned antigens is dominatedby the spatial tolerance of antibodies, Nature Nanotechnology, 14(2),184–190.

Shaw, A., Lundin, A., Petrova, E., F€ord}os, F., Benson, E., Al-Amin, A., …Teixeira, A. (2014). Spatial control of membrane receptor functionusing ligand nanocalipers. Nature Methods, 11(8), 841–846.

23. DNA is not merely the secretof life: semantomorphic chemistryin advanced materials

Nadrian C SeemanNew York University, New York, NY 10003, USA

We build branched DNA species that can be joined usingWatson-Crick base pairing to produce N-connected objectsand lattices. We have used ligation to construct DNA topo-logical targets, such as knots, polyhedral catenanes,Borromean rings and a Solomon’s knot.

Nanorobotics is a key area of application. We have maderobust 2-state and 3-state sequence-dependent program-mable devices and bipedal walkers. We have constructed 2-dimensional DNA arrays with designed patterns from manydifferent motifs. We have used DNA scaffolding to organizeactive DNA components. We have used pairs of 2-state devi-ces to capture a variety of different DNA targets. We haveconstructed a molecular assembly line using a DNA origamilayer and three 2-state devices, so that there are eight differ-ent states represented by their arrangements. We have dem-onstrated that all eight products can be built from thissystem. Recently, we connected the nanoscale with themicroscale using DNA origami.

We have self-assembled a 3D crystalline array andreported its crystal structure to 4 Å resolution. We can usecrystals with two molecules in the crystallographic repeat tocontrol the color of the crystals. Rational design of intermo-lecular contacts has enabled us to improve crystal resolutionto better than 3Å. We can now do strand displacement inthe crystals to change their color, thereby making a 3D-based molecular machine; we can visualize the presence ofthe machine by X-ray diffraction.

The use of DNA to organize other molecules is central toits utility. Earlier, we made 2D checkerboard arrays of metal-lic nanoparticles, and have now organized gold particles in3D. Recently, we have ordered triplex components and asemiconductor within the lattice. Thus, structural DNA nano-technology has fulfilled its initial goal of controlling theinternal structure of macroscopic constructs in three dimen-sions. A new era in nanoscale control awaits us.

24. A novel indoline scaffold-basedantibacterial compound designingand pharmacological evaluation usingchemoinformatics approach

Aarushi Singh and Ramesh ChandraDrug Discovery & Development Laboratory, Departmentof Chemistry, University of Delhi, Delhi 110007, India

[email protected]

Antibiotic resistance is not only a global public health threatbut also a huge economic burden to our society thaturgently needs to be addressed by improved antibiotics andcontinuing development of novel molecules to treat resistantbacterial infections (Kim et al., 2008; Kaplancikli et al., 2007).Nowadays combination therapies offer a competentapproach to counteract antibiotic resistance in bacteria(Singh et al., 2016, 2017). Better knowledge of mechanismsof antibiotic resistance has led to the finding of new alterna-tives to antibiotic therapy. Hence, in this article, we report anovel series of indoline derivatives and their computational

Figure. The Central Concept of Structural DNA Nanotechnology: CombineBranched DNA with Sticky Ends to Make Objects, Lattices and Devices.


study as potent antimicrobials. The present study investi-gates the indoline based derived library interaction withDNA gyrase B enzyme to be used as a potential antimicrobialdrug. Computational approaches were employed to carry outthe molecular interactions and pharmacological studies. Inthis study, we have compared indoline with its derivativesand have found that compound 13 resulted in the strongbinding with the highest score (% 9.02 kcal/mol) in thedesigned library where indoline showed (% 6.43 kcal/mol).Furthermore, molecular dynamics simulation run also con-firmed the strongest interaction of a compound and targetprotein with less RMSD and RMSF deviation of the complex.Notably, the compound was also found to possess the goodpharmacological properties and pharmacokinetic properties.

References

Kaplancikli, Z. A., Turan-Zitouni, G., Ozdemir, A., Revial, G., & Guven, K.(2007). Phosphorus, Sulfur, and Silicon and the Related Elements, 182(4),749–764.

Kim, S.-W., Kuti, J. L., & Nicolau, D. P. (2008). Inhaled antimicrobial therapiesfor respiratory infections. Current Infectious Disease Reports, 10(1), 29–36.

Singh, V. K., Kumar, N., & Chandra, R. (2017). Structural insights ofinduced pluripotent stem cell regulatory factors Oct4 and its inter-action with Sox2 and Fgf4 gene. Advances in Biochemistry andBiotechnology, J119.

Singh, V. K., Kumar, N., Kalsan, M., Saini, A., & Chandra, R. A. (2016).Novel peptide thrombopoietin mimetic designing and optimization

using computational approach. Frontiers in Bioengineering andBiotechnology, 4(69)

25. Discovery of penicillin bindingproteins (PBPs) inhibitors by blendingvirtual screening, molecular dockingand simulation studies

Mayurpankhi Buragohaina, Abha Vashishthab andSurabhi JoharibaCentre for Biotechnology and Bioinformatics Dibrugarh University,Dibrugarh, Assam, 786001, India; bSchool of Biosciences, Instituteof Management Sciences, University Courses, Ghaziabad, UttarPradesh, 201002, India [email protected]

KEYWORDS Methicillin; molecular docking; penicillin binding proteins;resistance; triazine simulations

An integrated protocol of virtual screening involving molecu-lar docking, pharmacophore probing and simulations wasestablished to identify small novel molecules targeting cru-cial residues involved in the penicillin binding proteins(PBPs) involved in bacterial cell wall biosynthesis. An excel-lent approach was made utilizing ligand and structure-basedpharmacophore to identify common features and structure-


based docking with respect to PBPs in leading to the devel-opment of novel inhibitors possessing new scaffolds. To ourdelight, multiple-substituted triazine derivatives series bear-ing a novel scaffold, of which structure is remarkably differ-ent from the existing b-lactam inhibitors, were designed. Astructure-based pharmacophore mapping was developed toexplore the binding sites of PBPs which were taken into con-sideration. Subsequently, virtual screening, ADMET searcheswere at work to narrow down the proposed hits to be for-warded as a potential drug likes candidates. Further, thebinding patterns of the best-proposed hits were explored tounderstand the deeper insight for its structural optimizationby employing it on molecular dynamic simulations of 500ps.Selectivity profile for the most promising candidates wasstudied, revealing significantly C series molecules C15 wasfound to be the most potent compounds among designedlibrary. The proposed hits can be forwarded for further studyagainst PBPs involved in bacterial peptidoglycan cell wallbiosynthesis.

References

Desai, N., Makwana, A., & Rajpara, K. (2016). Synthesis and study of 1,3,5-triazine based thiazole derivatives as antimicrobial agents. Journal ofSaudi Chemical Society, 20, S334–S341.

Gonzales, P., Pesesky, M., Bouley, R., Ballard, A., Biddy, B., Suckow, M., …Dantas, G. (2015). Synergistic, collaterally sensitive b-lactam combina-tions suppress resistance in MRSA. Nature Chemical Biology, 11(11),855–861.

26. DNA topoisomerases as targetsfor antibacterial chemotherapy

Anthony MaxwellDepartment of Biological Chemistry, John Innes Centre, NorwichResearch Park, Norwich, NR4 7UH, UK [email protected]

DNA topoisomerases are enzymes that catalyze changes inDNA topology (Bates & Maxwell, 2005). There are two types,I and II, differentiated by whether they catalyze reactions viasingle- or double-stranded breaks in DNA. They are essentialin all cells, having key roles in DNA replication, transcriptionand recombination. All topoisomerases are able to relaxsupercoiled DNA, but DNA gyrase, essential in all bacteria,can also introduce negative supercoils in a reaction coupledto ATP hydrolysis (Collin, Karkare, & Maxwell, 2011). Due totheir essential nature and the fact that they stabilize single-or double-stranded breaks in DNA, topoisomerases havebecome key drug targets for both antibacterial and anti-can-cer chemotherapy (Collin, Karkare, & Maxwell, 2011; Maxwell,Bush, Germe, & McKie, 2018; Pommier, Leo, Zhang, &Marchand, 2010) (Figure 1).

Building on structural and mechanistic data, we havefocused on antibiotics targeted to bacterial gyrase.Fluoroquinolone drugs (e.g., ciprofloxacin) have been highly

successful. Their mechanism involves stabilization of a pro-tein-linked transient double-strand break in DNA, which canbe lethal. However, resistance to fluoroquinolones is signifi-cant and alternative compounds are needed. Although ami-nocoumarin natural products (e.g., novobiocin) are potentialalternatives, these compounds have not achieved significantclinical success. Working with pharma companies we haveinvestigated other compounds that stabilize the gyrase-DNAcleavage complex, e.g., IPYs (Germe et al., 2018; Jeannotet al., 2018) and thiophenes (Chan et al., 2017) (Figure 1).The potential of these and other compounds as future antibi-otics will be discussed.

References

Bates, A. D., & Maxwell, A. (2005). DNA Topology. Oxford: OxfordUniversity Press.

Chan, P. F., Germe, T., Bax, B. D., Huang, J., Thalji, R. K., Bacque, E., …Ding, X. (2017). Thiophene antibacterials that allosterically stabilizeDNA-cleavage complexes with DNA gyrase. Proceedings of theNational Academy of Sciences of the United States of America, 114(22),E4492–E4500.

Collin, F., Karkare, S., & Maxwell, A. (2011). Exploiting bacterial DNAgyrase as a drug target: Current state and perspectives. AppliedMicrobiology and Biotechnology, 92(3), 479–497.

Germe, T., Voros, J., Jeannot, F., Taillier, T., Stavenger, R. A., Bacque, E.,… Bax, B. D. (2018). A new class of antibacterials, the imidazopyrazi-nones, reveal structural transitions involved in DNA gyrase poisoningand mechanisms of resistance. Nucleic Acids Research, 46(8),4114–4128.

Jeannot, F., Taillier, T., Despeyroux, P., Renard, S., Rey, A., Mourez, M., …Versluys, S. (2018). Imidazopyrazinones (IPYs): Non-quinolone bacterialtopoisomerase inhibitors showing partial cross-resistance with quino-lones. Journal of Medicinal Chemistry, 61(8), 3565–3581.

Maxwell, A., Bush, N. G., Germe, T., & McKie, S. J. (2018). In Fong, I. W., D.Shlaes & K. Drlica (eds.), Antimicrobial resistance and implications forthe 21st century. Springer.

Pommier, Y., Leo, E., Zhang, H., & Marchand, C. (2010). DNA topoisomer-ases and their poisoning by anticancer and antibacterial drugs.Chemistry & Biology, 17(5), 421–433.

27. Exploring peptidoglycanbiosynthesis and it’s inhibition

Adrian Lloyd, Dom Bellini, Hector Newman andChristopher DowsonSchool of Life Sciences, University of Warwick, Coventry, CV47AJ,UK [email protected]

Figure 1. A cartoon of a truncated gyrase-DNA complex highlighting the sitesof action of antibiotics.


Penicillin and the wider family of beta-lactams haveremained the single most important family of antibioticssince their introduction in the early 1940s. Unfortunatelythey are becoming less effective due to the emergence ofresistance that now has an impact globally (E. P. Center forDisease Dynamics, 2015). Predominantly, resistance is medi-ated by reducing the target drug concentration by catalysingtheir inactivation by beta-lactamases (Bush, 2015; Bush andBradford, 2016; Drawz, Papp-Wallace, & Bonomo, 2014;Ehmann et al., 2012; Lahiri et al., 2013), by increasing efflux, orreducing permeation (in Gram negative bacteria). The periplas-mic/extracytoplasmic targets of penicillin are a family ofenzymes with a highly conserved catalytic activity involved inthe final stage of bacterial cell wall biosynthesis: cross-linkingof the structural polymer peptidoglycan (PG). These enzymes,named after their ability to bind penicillin, rather than theircatalytic activity are called penicillin-binding proteins (PBPs)(Frere & Page, 2014; Sauvage, Kerff, Terrak, Ayala, & Charlier,2008; Schweizer et al., 2017). Surprisingly, given the pivotalimportance of PBPs, we still have a very incomplete picture ofhow PBPs interact with their natural substrates, precisely whatthese substrates are, exactly how beta-lactam antibiotics inter-fere with this process and the response of bacteria to generatea further resistance mechanism, PBP-mediated resistance.

Here we discuss recent advances in the development ofnew reagents assays and structures that are helping todevelop a rigorous biochemical structure functional analysisof how the targets of penicillin work, including a full kineticcharacterization, and the interplay between enzyme sub-strate and inhibitor.

References

Bush, K. (2015). A resurgence of b-lactamase inhibitor combinationseffective against multidrug-resistant Gram-negative pathogens.International Journal of Antimicrobial Agents, 46(5), 483–493.

Bush, K., & Bradford, P. A. (2016). Cold Spring Harbor Perspectives inMedicine, 6

Drawz, S. M., Papp-Wallace, K. M., & Bonomo, R. A. (2014). New b-lacta-mase inhibitors: A therapeutic renaissance in an MDR world.Antimicrobial Agents and Chemotherapy 58(4), 1835–1846.

Ehmann, D. E., Jahi"c, H., Ross, P. L., Gu, R. F., Hu, J., Kern, G. … Fisher,S. L., (2012). Avibactam is a covalent, reversible, non-b-lactam b-lacta-mase inhibitor. Proceedings of the National Academy of Sciences of theUnited States of America, 109(29), 11663–11668.

E. P. Center for Disease Dynamics 2015 Journal (2015).Frere, J. M., & Page, M. G. (2014). Penicillin-binding proteins: Evergreen

drug targets. Current Opinion in Pharmacology, 18, 112–119.Lahiri, S. D., Mangani, S., Durand-Reville, T., Benvenuti, M., Luca, F. D.,

Sanyal, G., & Docquier, J.-D. (2013). Structural insight into potentbroad-spectrum inhibition with reversible recyclization mechanism:Avibactam in complex with CTX-M-15 and Pseudomonas aeruginosaAmpC b-Lactamases. Antimicrobial Agents and Chemotherapy, 57(6),2496–2505. doi: 10.1128/AAC.02247-12.

Sauvage, E., Kerff, F., Terrak, M., Ayala, J. A., & Charlier, P. (2008). Thepenicillin-binding proteins: Structure and role in peptidoglycan bio-synthesis. FEMS Microbiology Reviews, 32(2), 234–258.

Schweizer, I., Bl€attner, S., Maurer, P., Peters, K., Vollmer, D., Vollmer, W.… Denapaite, D., (2017). New aspects of the interplay between peni-cillin binding proteins, murM , and the two-component system ciarhof penicillin-resistant Streptococcus pneumoniae Serotype 19A iso-lates from Hungary. Antimicrobial Agents and Chemotherapy, 61(7),doi: 10.1128/AAC.00414-17.

28. How bacteria and cancer cellsregulate mutagenesis and their abilityto evolve

Susan M. RosenbergBaylor College of Medicine, Houston, TX, USA

Our concept of genomes is changing from one in whichthe DNA sequence is passed faithfully to future generationsto another in which genomes are plastic and responsive toenvironmental changes. In contrast with classical assump-tions that mutations occur purely stochastically at constant,gradual rates, microbes, plants, flies and human cancer cellspossess mechanisms of mutagenesis that are upregulatedby stress responses. These generate transient, genetic-diver-sity bursts that can propel evolution, including evolution ofinfectious disease and cancers, specifically when cells arepoorly adapted to their environments—that is, whenstressed. Emerging molecular mechanisms of stress-indu-cible mutagenesis vary but share common components thathighlight the non-randomness of mutation: (1) regulation ofmutagenesis in time by cellular stress responses, which pro-mote mutations when cells are poorly adapted to theirenvironments—when stressed; (2) limitation of mutagenesisin genomic space causing mutation hotspots and clusters,which may both target specific genomic regions and allowconcerted evolution (evolution requiring multiple muta-tions). This presentation will focus on the molecular mech-anism of stress-inducible mutagenic DNA break repair in E.coli as a model for stress-inducible mutagenesis that pro-pels infectious disease, antibiotic resistance and other evo-lution. We consider its regulation by stress responses,demonstrate its formation of mutation hotspots near DNAbreaks, and our discovery of a large gene network thatunderlies mutagenic break repair, most of which functionsin stress sensing and signaling. We also show that muta-genesis is induced by the antibiotic ciprofloxacin, causingresistance to other antibiotics, and demonstrate the stress-response-dependent mutagenesis mechanism. Regulation ofmutagenesis in time and genomic space may accelerateevolution including evolution of pathogens, cancers, anddrug resistance.

29. Identifying and targetingthe Achilles heel of drugresistant bacteria

Nagasuma ChandraIndian institute of Science, Bangalore, India

The emergence of drug resistant strains of M. tuberculosisposes a major threat to public health, warranting urgentattention to the problem of tackling antimicrobial resistance.A number of studies have sought to understand the causes


of drug resistance, and have led to the identification of sev-eral mechanisms including mutations in key targets, upregu-lation of drug efflux pumps, etc. Yet, there is nounderstanding of which mechanisms are operative in a givencondition, whether microbes explore multiple mechanismssimultaneously, or if such mechanisms are influenced byeach other and lead to alterations in the cell in a synchron-ized manner. Toward this, an understanding of the globalmechanisms leading to resistance becomes necessary. This inturn will provide a basis to develop novel therapeutic strat-egies for tackling drug resistance.

To address this, we adopt a systems level analysis of thebiomolecular networks associated with the emergence orsustenance of drug-resistant phenotypes, from which wedecipher cellular alterations in an unbiased manner andidentify emergent vulnerabilities in resistant bacilli. We inte-grate genomic, transcriptomic and phenotypic data from themodel system M. smegmatis and generate an integrated gen-ome-scale response network, and employ network-miningapproaches to identify the highest differential activitiesoperative in the drug-resistant strain. The corresponding pro-teins are seen to form a well-connected orchestrated subnet,providing insights into newly emerged vulnerabilities in theresistant bacilli. The networks suggest that multiple mecha-nisms are at play simultaneously to overcome the drugstress, of which response to oxidative stress predominates.Through targeted screening we discover that the resistantbacilli exhibit collateral sensitivity to several compounds thatblock antioxidant responses. We then test the shortlistedcompounds against M. tuberculosis and three different drug-resistant clinical isolates and study the mechanistic basis ofthe efficacies of these compounds. Three of the tested com-pounds were able to reverse resistance in mycobacteria. Oneof the identified compounds shows high promise as a keycomponent of a re-purposable drug combination for treatingdrug-resistant tuberculosis.

30. Interactions of b-lactamases withantibiotics and inhibitors

Catherine L. Tookea,b, Philip Hinchliffea, RamyaSalimraja, Karina Calvopi~naa, Viivi H.A. Hirvonenb,Matthew B. Avisona, Marc W. van der Kampb,c,Adrian J. Mulhollandb and James SpenceraaSchool of Cellular and Molecular Medicine, University of Bristol,Bristol BS8 1TD, UK; bSchool of Chemistry, University of Bristol,Bristol BS8 1TS, UK; cSchool of Biochemistry, University of Bristol,Bristol BS8 1TD, UK [email protected]

Beta-lactams remain the single most important antibioticclass. Beta-lactamases are hydrolytic enzymes that inactivatethe antibiotic by degrading the scissile amide bond of thefour-membered beta-lactam ring, and are the most preva-lent resistance mechanism in Gram-negative bacterial patho-gens responsible for a variety of opportunistic infections. Todate more than 4000 beta-lactamases have been identifiedin environmental and clinical bacterial strains. These

enzymes vary widely in their activity against the differentbeta-lactam classes (principally penicillins, cephalosporinsand carbapenems) and their susceptibility towards inhibitorsthat include beta-lactam- (clavulanic acid) and non-beta-lac-tam (diazabicyclooctane; boronate) based agents. Beta-lacta-mases also divide into two groups: active-site serineenzymes (classes A, C and D) and zinc metallo-enzymes(class B) that differ fundamentally in structureand mechanism.

Our research focuses on interactions of beta-lactamaseswith carbapenems, the most recently introduced beta-lac-tams and key antibiotics for infections by Gram-negativebacteria. Carbapenems escape hydrolysis by most serinebeta-lactamases as the 6a-hydroxyethyl substituent and tau-tomerization within the fused pyrroline ring system aretogether proposed to retard breakdown of the covalentacylenzyme intermediate; carbapenem hydrolysis (carbape-nemase activity) is restricted to a select few serine enzymes.In contrast, metallo-beta-lactamases efficiently hydrolyze car-bapenems. We have investigated carbapenem hydrolysis byboth serine carbapenemases and metallo-beta-lactamases,combining high-resolution crystallographic methods andhigh-level computational (molecular dynamics and quantummechanics/molecular mechanics (QM/MM)) approaches toseek to identify the requirements for hydrolysis of thesesubstrates. I will further describe the interactions of bothserine and metallo-beta-lactamases with a range of inhibi-tors, extending from compounds in clinical use to experi-mental compound series aimed at expanding inhibition toinclude the metallo-beta-lactamases against which currentagents are ineffective.

31. Kinetic and moleculardeterminants of antibioticpermeation barriers

Helen I. ZgurskayaaDepartment of Chemistry and Biochemistry, University ofOklahoma, Norman, OK 73019, USA

The permeability barrier of Gram-negative cell envelopes isthe major obstacle in the discovery and development of newantibiotics. In Gram-negative bacteria, these difficulties areexacerbated by the synergistic interaction between two bio-chemically distinct phenomena, the low permeability of theouter membrane and active multidrug efflux. We developedan approach to separate the contributions of the two mecha-nisms in the activities of antibiotics and applied it in the dis-covery of efflux pump inhibitors and analyses ofstructure–activity relationships for active efflux and outermembrane permeation in different Gram-negative patho-gens. This presentation will be focused on species- and con-text-dependent interplays between active drug efflux anddrug permeation and how they affect antibacterial activitiesof antibiotics, substrate specificities of efflux pumps andefflux inhibitors.


32. A comparative computationalstudy for deamination of trans andgauche conformers ofphenylethylamine in presenceof lumiflavin

Subrata Dasgupta, Soumita Mukherjee and B.P.MukhopadhyayDepartment of Chemistry, National Institute of Technology,Durgapur, Durgapur 713209, West Bangal, India

[email protected]

The deamination of phenylethylamine (PEA) and norepineph-rine (NOR) to corresponding aldehyde and ammonia by theenzyme human Monoamine oxidase (hMAO) is one of themost important concerns in neurobiochemistry due to itsinvolvement in several neurological diseases and complica-tions. The enzyme has two isoforms with 70% sequence simi-larity. Our earlier studies with dopamine-hMAO B showedpersistence of trans form at the active site cavity (Dasgupta,Mukherjee, Mukhopadhyay, Banerjee, & Mishra, 2018)whereas MD —simulation followed by DFT studies forphenylethylamine(PEA)-hMAO B complex reveals existence ofboth trans and gauche conformation for protonated PEA(Dasgupta, Mukherjee, & Mukhopadhyay, 2018). In this workconcerted hydride transfer mechanism for trans and gaucheconformers of neutral-PEA with lumiflavin moiety (truncatedprosthetic group involved during catalysis) have been per-formed by density functional theory (DFT) methods usingB3LYP/DFT-D3 functional with standard split valance basis set6-31 g. This computational work provides the first evidencethat could support the easier feasibility of deamination ofgauche conformer of hMAO substrates over trans-substrate.The activation energy barriers for trans and gauche PEA arecompared, in which gauche conformation is observed to bepreferred over trans conformer. The result indicates that thedeamination of the neurotransmitters may depend on the con-formational preference of phenylethylamine (Figure 1).

Funding

This research has been financially supported by ICMR(Project No. ISRM/11 (25)/2017), Government of India.

References

Dasgupta, S., Mukherjee, S., & Mukhopadhyay, B. P. (2018). Recognitionof trans and gauche phenylethylamine conformers in the active siteof human monoamine oxidase B: A MD-simulation and DFT studies.Computational and Theoretical Chemistry, 1127, 44–51.

Dasgupta, S., Mukherjee, S., Mukhopadhyay, B. P., Banerjee, A., & Mishra,D. K. (2018). Recognition dynamics of dopamine to humanMonoamine oxidase B: Role of Leu171/Gln206 and conserved watermolecules in the active site cavity. Journal of Biomolecular Structureand Dynamics, 36(6), 1439–1462.

33. A comprehensive automatedcomputer-aided discovery pipelinefrom genomes to hit molecules

Ruchika Bhata,b, Rahul Kaushikb,c, Ankita Singhb,Debarati DasGuptaa,b, Abhilash Jayaraja,b, AnjaliSonia,b, Ashutosh Shandilyaa,b, Vandana Shekharb,Shashank Shekharb and B. Jayarama,b,c

aDepartment of Chemistry, Indian Institute of Technology, Delhi,Hauz Khas, New Delhi, 110016, India; bSupercomputing Facility forBioinformatics & Computational Biology, Indian Institute ofTechnology, Delhi, Hauz Khas, New Delhi 110016, IndiaandcKusuma School of Biological Sciences, Indian Institute ofTechnology, Delhi, Hauz Khas, New Delhi, 110016, India

[email protected]; [email protected]

Big data generation through sequencing of genomes andproteomes has led to over 2800 whole genomes and " 84million protein sequences, however, utilization of these datato generate lead molecules for curing diseases remains achallenge. We propose here, Dhanvantari, a comprehensivesoftware suite which automates the computational journeyfrom genome to hit molecules via its various genomics, pro-teomics and drug designing modules with possible entry atany module. The proposed software suite offers new oppor-tunities and insights to ‘genome-based’ drug discovery alongwith classical structure-based approaches to discover newdrug-like molecules. The pipeline helps in exploring newpotential drug targets from genomic/proteomic data whichwere earlier inaccessible and helps to find novel hits viascreening against million compounds or natural products orFDA approved drugs or even customized molecule libraries.

Figure 1.


Case studies on Hepatitis B Virus and Hepatitis A Virusagainst their druggable proteins via this pipeline have led topotent and novel inhibitors with low micro molar rangeinhibitions in in vitro studies. The entire protocol proposed inDhanvantari requires " 6–12 h, however, individual steps getcompleted within few minutes. The software suite is madefreely accessible as an online resource at http://www.scfbio-iitd.res.in/software/dhanvantari_new/Home.html with no add-itional dependencies. Presently, there is no fully-automatedopen source similar to Dhanvantari which can mine the infor-mation about the potential drug like molecules from thegenome as well as proteome information (Figure 1).

References

Soni, A., Pandey, K. M., Ray, P., & Jayaram, B. (2013). Genomes to hits insilico—a country path today, a highway tomorrow: A case study ofChikungunya. Current Pharmaceutical Design, 19(26), 4687–4700.

34. A fluoroamine derivative ofepigallocatechin gallate exhibits both,anti-cancer and anti-tuberculosisproperties. Insights from in silico andin vitro studies

Samarendra Narayanan, Ramesh andK.V. Dipti MothaySchool of Sciences, Jain University, Jayanagar, Bangalore 560011,India


Epigallocatechin gallate (EGCG), has been reported to haveboth anti-cancer and anti- mycobacterial properties.Fluorinated compounds are known to inhibit both TB andcancer (Hoagland et al., 2016). Few derivatives of EGCG weredrawn structurally and subsequently chemically synthesized.A derivative with a fluorine and amine moiety was analyzedfor inhibitory properties against (M. tb) and cancer using insilico, and in vitro approaches. In silico approach includeddocking of the optimized ligand to the enoyl reductase (ER)

receptor of M. tb using AutoDock (v 4.0) and Hex. MolecularDynamic (MD) simulation of this docked receptor-ligandcomplex using Gromacs (v.4.2) was then conducted.Fluoroamine derivative of EGCG inhibited Mycobacteriumsmegmatis, in silico (dock energy -10.9 kcal mol-1), in vitroand also inhibited the tyrosine kinase (TK) receptor Her2 andtaxanes (TXL) microtubule receptor of cancer cells. The fluo-roamine derivative also showed better inhibition ofMycobacterium smegmatis than EGCG based on these studies.Further, MD simulation of the fluoroamine derivative sub-stantiated the docking results, since the receptor ligand com-plex showed greater stability than the receptor protein after50 ns of MD simulation as evidenced from the RMSD, RMSFand energy profiles (Potential Energy, Kinetic Energy andTotal Energy) in line with observations of Gholami andBordbar (2017). The anti-cancer properties of the fluoroaminederivative evaluated in vitro in the Jurkat human leukemiacell line, showed almost a tenfold better inhibition thanEGCG as reported in the literature. Taken together, thesefindings hold much promise for further study of the anti-TBand anti-cancer properties of the fluoroamine derivative.

References

Gholami, S., & Bordbar, A. K. (2017). Putative binding sites of dopamine andarachidonoyl dopamine to beta lactoglobulin: A molecular docking andmolecular dynamics study. Physical Chemistry Research, 5(2), 205–219.

Hoagland, D. T., Liu, J., Lee, R. B., & Lee, R. E. (2016). New agents for thetreatment of drug resistant Mycobacterium tuberculosis. AdvancedDrug Delivery Reviews, 102, 55–72.

35. A new approach to analyzerotavirus transport mechanism inporous media by molecular modelingand molecular dynamics methods

Elena M. Alvareda Migliaroa,b , Jorge Canterob,Fernando L"opez Tortc, Mat"ıas Victoriac,Margot Paulino Zuninib, Rodney Colinac andPablo A. Gamazoa

Figure 1.


aWater Department, CENUR, Universidad de la Rep"ublica, Salto,Uruguay; bCenter for Structural Bioinformatics, DETEMA - Facultadde Qu"ımica, Universidad de la Rep"ublica, Montevideo, Uruguay;cVirology Laboratory, CENUR, Universidad de la Rep"ublica, Salto,Uruguay [email protected]

It has always been believed that groundwater was a safesource, due to the ‘naturalfilter’ which takes place in porousmedia. However, groundwater could be polluted with micro-organism or chemical products from wastewater from septictanks or leaking sewage pipes. In most cases people drinkthis underground water without treatment and this could bethe source of viral gastroenteritis outbreaks in Salto city,located on the Northwestern region of Uruguay. The evalu-ation of groundwater pollution in Salto by means of thepresence and the incidence of human gastroenteric viruseslike Rotavirus A has been studied recently. Furthermore, tounderstand the transport mechanisms of this virus, a studyof the kinetic aspects of the adsorption isotherm mecha-nisms by means of column assays have been performed byour group. These results showed that Rotavirus A wasdetected in the Salto aquifer, and similar concentrations inSalto sewer effluent were measured. The aim of this studywas to understand the main molecular characteristics respon-sible for the Rotavirus A transport mechanism from naturalsoil-water systems that could affect it adsorption and inacti-vation in porous media. We proposed: (1) A 100 ns explicitsolvent MD simulation of the crystal structure of the rhesusrotavirus VP4 sialic acid binding domain in complex with 2-O-methyl-alpha-D-N-acetyl neuraminic acid (PDBid: 1KQR)with NAMD program. (2) A study of the interaction energiesof solvent-protein. (3) An analysis of the interactionsbetween the VP4 virus protein with an inorganic silica mem-brane model by Stereed Molecular Dynamic (SMD) to explainthe structural and energetic features of protein membraneassociation. We obtained a validated model of VP4 with thesialic acid ligand and we discussed the molecular interactionsof Rotavirus A in an inorganic environment (Figure 1).

Funding

CSIC and National Research and Innovation Agency ANII(FMV_2_2011_1_6927 project) have supported this research.

ORCID

Elena M. Alvareda Migliaro http://orcid.org/0000-0002-6065-5741

References

Gamazo, P., Victoria, M., Schijven, J. F., Alvareda, E., Lopez, T. L. F.,Ramos, J., … Colina, R. (2018). Evaluation of bacterial contaminationas an indicator of viral contamination in a sedimentary aquifer inUruguay. Food and Environmental Virology, 1–11.

Gamazo, P., Victoria, M., Schijven, J. F., Alvareda, E., Tort L. F, L., Ramos,J., … Colina, R. (2016). Comparison of rotavirus and norovirus trans-port in standardised and natural soil-water systems, Abstract H33P-06presented at 2016 Fall Meeting, AGU, San Francisco, Calif., 11-15 Dec.

Settembre, E. C., Chen, J. Z., Dormitzer, P. R., Grigorieff, N., & Harrison,S. C. (2011). Atomic model of an infectious rotavirus particle. TheEMBO Journal, 30(2), 408–416.

36. Evidence for the N-Terminalhypothesis for Alzheimer’s disease

Georges BelfortHoward P. Isermann Department of Chemical and BiologicalEngineering and the Center for Biotechnology andInterdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY12180, USA

Although the amyloid (abeta peptide, Ab) hypothesis is25 years old, is the dominant model of Alzheimer’s Disease(AD) pathogenesis, and currently guides the development ofpotential treatments, it is still controversial. One possible rea-son is a lack of a clear mechanistic path from the cleavageproducts of the amyloid precursor protein (APP) such as sol-uble Abmonomer and a series of recently discovered solublemolecular fragments to the deleterious effects on synapticform and function (Willem et al., 2015; Welzel et al., 2014).Both biophysical properties of these molecular entities andthe balance between production and clearance are consid-ered critical for AD pathogenesis. From a review of therecent literature including aggregation kinetics (Murray et al.,2016) and structural morphology (Murray et al., 2016), Abclearance (Murray et al., 2016), molecular simulations (Das,Murray, & Belfort, 2015), long-term potentiation measure-ments with inhibition binding, and the binding of a commer-cial monoclonal antibody, aducanumab, we hypothesize that

Figure 1.


the N-terminal domains of neurotoxic Ab oligomers areimplicated in causing the disease. We call this the ‘N-Terminal Hypothesis for AD’. The supporting evidence relatedto this hypothesis includes the discovery of the following: (i)The first protective mutation of APP (A673T) or Ab (A2T)against AD reduces BACE1 cleavage at the b-secretase pos-ition (N-terminus of Ab) resulting in a drop in Ab concentra-tion of about 40% (Jonsson et al., 2012). (ii) A causativemutation at the same location (A673V) or Ab (A2V) againstAD results in an increase in Ab concentration of about 100%.(iii) The A2T and A2V mutants of Ab1-42 increase the aggre-gation lag time prior to the onset of fibril formation com-pared with wild type Ab1-42 by a factor of " 1.5 and " 8,respectively (Murray et al., 2016). (iv) Aggregate morphologyof the N-terminal mutants (A2T and A2V) is altered comparedwith wild type Ab1-42 (Murray et al., 2016). (v) Moleculardynamic (MD) simulations show the importance of N-ter-minus on monomer folding and lowering of the formation ofneurotoxic b-hairpin structures (Das et al., 2015). (vi) Long-term potentiation (LTP) deficit, which correlates with memoryand learning, is reduced for the A2T Ab1-42 mutant, in com-parison with the wild type and A2V Ab1-42 mutant (Murrayet al., 2016). (vii) Blocking the N-terminus with sequence spe-cific antibodies prevents LTP deficit whereas blocking thehydrophobic central core and C-terminus have minimaleffects. (viii) Fragments of APP induce LTP deficits only whenthe N-terminus is included in the fragment (Willem et al.,2015). (ix) A promising recombinant human monoclonal anti-body, aducanumab, binds to the N-terminus of Ab oligomers.(x) Recently, reported experimental structures also show aflexible/exposed N-terminal region (Ab1-14) in the diseaserelevant Ab1-42 fibril (Walti et al., 2016). Additionally, sincemonomers are not neurotoxic while dimers, trimers etc. are,this suggests that at least two N-termini are necessary forLTP deficit induction. It then follows that the mechanism oftoxicity could be due to multivalent interactions betweenthe N-termini (& 2) of Ab oligomers with glutamate N-methylD-aspartate receptors. Unpublished MD simulations demon-strate increased flexibility of the protective versus the causa-tive variants for N-termini in dimers. Taken together, thesecollective findings strongly suggest that the N-terminus ofAb oligomers could be causative for AD.

References

Das, P., Murray, B., & Belfort, G. (2015). Alzheimer’s protective A2T muta-tion changes the conformational landscape of the Ab 1–42 monomerdifferently than does the A2V mutation. Biophysical Journal, 108(3),738–747.

Jonsson, T., Atwal, J. K., Steinberg, S., Snaedal, J., Jonsson, P. V.,Bjornsson, S., … Stefansson, K. (2012). A mutation in APP protectsagainst Alzheimer’s disease and age-related cognitive decline. Nature,488(7409), 96–99.

Murray, B., Sorci, M., Rosenthal, J., Lippens, J., Isaacson, D., Das, P., …Belfort, G. (2016). A2T and A2V Ab Peptides exhibit different aggrega-tion kinetics, morphology, structure and LTP inhibition. Proteins:Structure, Function, and Bioinformatics, 84(4), 488–500.

Walti, M. A., Ravotti, F., Arai, H., Glabe, C. G., Wall, J. S., Bockmann, A., …Riek, R. (2016). Atomic-resolution structure of a disease-relevantAbeta(1-42) amyloid fibril. Proceedings of the National Academy ofSciences of the United States of America.

Welzel, A. T., Maggio, J. E., Shankar, G. M., Walker, D. E., Ostaszewski,B. L., Li, S., … Walsh, D. M. (2014). Secreted amyloid b-proteins in acell culture model include N-terminally extended peptides that impairsynaptic plasticity. Biochemistry, 53(24), 3908–3921.

Willem, M., Tahirovic, S., Busche, M. A., Ovsepian, S. V., Chafai, M.,Kootar, S., … Daria, A. (2015). g-Secretase processing of APP inhib-its neuronal activity in the hippocampus. Nature, 526(7573),443–447.

37. Calculation of protein–ligand andprotein–protein binding affinitiesusing free energy perturbation theory

Richard A. FriesnerDepartment of Chemistry, Columbia University, 3000 Broadway,New York, NY 10027, USA [email protected]

Over the past several years, free-energy perturbation (FEP)molecular dynamics simulation methods have been shownto be capable of achieving useful prediction of protein–li-gand binding affinities, with a root-mean-square error onthe order of 1 kcal/mole. Recently, results of similar qualityhave been obtained for protein–protein binding affinitiesas well. At present, high-resolution structural data arerequired as a starting point for these calculations to suc-ceed. We will outline the methodological advances ena-bling reliable and accurate results to be obtained, anddiscuss applications to structure-based drug discovery.Finally, a brief discussion of new induced fit docking meth-ods, which will enable FEP to be effectively utilized in theabsence of a high-resolution crystal structure, willbe presented.

38. Cancer vaccine designing andoptimization using combinedimmunoinformatics and moleculardynamics simulation approach

Neeraj Kumar and Ramesh ChandraDepartment of Chemistry, University of Delhi, Delhi 110007, India


Cancer immunotherapy comes out to be an outstandingtreatment for cancer treatment and prevention. Cancer vac-cines are responsible for generating immune response toeliminate cancer cells, being given to cure cancer. In silicotechniques has brought efficient way to develop vaccines, asthese take less time to predict potential peptide epitopenonamers (Buhrman et al., 2013; Neeraj et al., 2017). In thisstudy, we have designed a potent vaccine candidate for can-cer using immunoinformatics approaches. Cytotoxic T-lymphocyte epitope as a Peptide vaccine was designed toelicit a desirable immune response against cancers, overex-pressing melanoma-associated antigen-A11 (MAGE-A11). Potent


epitope was predicted using reliable algorithms and validatedwith HLA-A!0201 and androgen receptor interaction (Dong et al2004; Henderson, Mossman, Nairn, & Cheever, 2005).

Our results showed a potent vaccine candidate ‘KIIDLVHLL’designed out of top ten epitope epitopes predicted using fivedifferent algorithms. Furthermore, epitope was validated withmolecular docking resulted in strong binding with HLA-A!0201 and androgen receptor with docking score -780.6 kcal/mol and -641.06 kcal/mol respectively. Lead epitope showedthe remarkable result with strong interactions (hydrogenbonds and hydrophobic interactions), the radius of gyrationscore of 23.0777 Å, world population coverage of 39.08% byimmune epitope database and TAP affinity IC50 value of2039.65 nm. This study paves the way to potential vaccine can-didate for the prevention of cancer (Figure 1).

References

Buhrman, J. D., & Slansky, J. E. (2013). Improving T cell responses tomodified peptides in tumor vaccines. Immunologic Research, 55(1–3),34–47.

Dong, H.-L., Li, Z.-S., Ye, J., Qu, P., Huang, Y.-Y., Wu, W., … Sui, Y.-F.(2004). Identification of HLA-A2-restricted CTL epitope encoded bythe MAGE-n gene of human hepatocellular carcinoma. Cancer Biology& Therapy, 3(9), 891–898.

Henderson, R. A., Mossman, S., Nairn, N., & Cheever, M. A. (2005). Cancervaccines and immunotherapies: Emerging perspectives. Vaccine,23(17/18), 2359–2362.

Neeraj, K., Chugh, H., Tomar, R., Tomar, V., Singh, V. K., & Chandra, R.(2017). Exploring the interplay between autoimmunity and cancer tofind the target therapeutic hotspots. Artificial Cells, Nanomedicine, andBiotechnology: An International Journal, 46 (4), 658–668. [10.1080/21691401.2017.1350188]

39. Carboline derivative compoundstargeting DNA: synthesis, binding andin vitro cytotoxicty on human cancercell lines

Kakali BhadraDepartment of Zoology, University of Kalyani, Kalyani, 741235,West Bangal, India [email protected]

KEYWORDS carboline derivative compounds; nucleic acid-small molecularbinding; FTIR; ITC; cell cytotoxicity; apoptosis

Three sets of carboline derived compounds were preparedby Pictet-Spengler cyclization. These tetrahydro b- and c-car-bolines have CF3 group with an additional amino alkyl chains(a- or d-position) and guanidine alkyl chains (a-position), ofvarying length. Structure–activity relationship of these mole-cules with calf thymus DNA was emphasized by fluorescence,ITC, FTIR and viscosity. Binding with DNA resulted in dra-matic enhancement and quenching in the fluorescence emis-sion. Gamma-carboline analogs showed maximum DNAbinding followed by beta-carboline compounds with aminoalkyl chain and least with guanidine alkyl chain compounds.It decreased with increasing chain length. The bindings wereentropically driven being more with guanidine alkyl chainanalogs. Site preference and mode of binding with partialintercalation and external binding was supported by FTIRand viscosity. Cytotoxic potencies of the compounds weretested on seven different cancer cell lines. The smallest alkylchain analog attached to gamma position, Comp3, showedmaximum cytotoxicity with GI50 6.2 mM, against HCT-116causing apoptosis, followed by the guanidine alkyl chaincompounds, but amino alkyl chain compounds to beta pos-ition showed poor cytotoxicity.

These results may be of prospective use in a frameworkto design novel carboline derivatives as antitumor drugs forimproved therapeutic applications in future.

Acknowledgment

I would like to acknowledge Prof. Valentine Nenajdenko and team fromMoscow State University, Moscow, for the synthesis of the compoundsand also grateful to DST-RFBR 2017-19 for funding of the research.

40. Antioxidant enzyme catalaseactivity of rat kidney cells nuclearfraction after the cisplatin andestradiol separate and joint action

Zhenya V. Yavroyan, Nune R. Hakobyan, Agapi G.Hovhannisyan, Gevorg A. Avagyan andEmil S. GevorgyanDepartment of Biophysics, Yerevan State University, Yerevan,Armenia [email protected]

Figure 1.


Cisplatin is a widely used and highly effective cancer chemo-therapeutic agent. Csplatin-induced cell death involves theactivation of multiple pathways of apoptoses as well as oxidantstress activation. These same pathways contribute to the cyto-toxic actions of cisplatin on tumor cells. However, it must benoted that cisplatin provided severe undesirable side effects. Themost common side effect of cisplatin is nephrotoxicity It is wellknown that oxidative stress has been recognized as an importantfactor that contributes to cisplatin nephrotoxicity. Nowadaysmany efforts have been made to employ drugs and other medi-caments as candidate adjuvants to cisplatin to minimize thisadverse influence. Steroid hormones such as sex hormones estra-diol and progesterone are presently considered as the best adju-vant to cisplatin, are able to prevent intoxication (Grott et al.,2013; Nematbakhsh et al., 2017). It is well known also, that cis-platin inhibits while estradiol promotes the activity of antioxidantenzymes. The aim of this research was to explore of catalase activ-ity in female rats kidney cells nuclear fraction after the cisplatinand estradiol separate and jointly action. As demonstrated ourresults cisplatin and estradiol provided opposite effects on cata-lase activity in case of separate application. In comparison withbaseline cisplatin alone action leads to decrease the catalaseactivity by 24%, whereas after the estradiol separate treatmentthe activity of this enzyme increases by 26%. The combinated useof cisplatin and estradiol leads to partial recovery of catalase activ-ity. In this case was recorded decrease of enzyme activity by 15%.Achieved results may be helpful for explaining of estradiol attenu-ating effects in case of its joint use with cisplatin.

References

Grott, M., Karakaya, S., Mayer, F., Baertling, F., Beyer, C., Kipp, M., & Kopp,H. G. (2013). Progesterone and estrogen prevent cisplatin-inducedapoptosis of lung cancer cells. Anticancer Research, 33(3), 791–800.

Nematbakhsh, M., Pezeshki, Z., & Eshraghi Jazi, F. (2017). Cisplatin-induced nephrotoxicity; protective supplements and gender differen-ces. The Asian Pacific Journal of Cancer Prevention, 18(2), 295–314.

41. Cisplatin and estradiol joint actionon quantities of rat brain chromatinphospholipids

Nune Hakobyan, Zhenya Yavroyan, Agapi G.Hovhannisyan and Emil S. GevorgyanDepartment of Biophysics, Yerevan State University, Yerevan,Armenia [email protected]

Nowadays, it is well known that nuclear lipids, including thechromatin bound ones, are important for regulating many essen-tial cellular processes such as DNA replication, transcription andgene expression. Recent advances demonstrated the involve-ment of nuclear lipids in remodeling of chromatin and epigen-etic regulation of gene expression. Furthermore, it was shownthat lipids of chromatin can regulate the chromatin structure viaalteration of some enzymes activity, which accomplished histonemodifications as well as inverse processes (Kuvichkin, 2016).

Our previous results showed the reliable changes in phos-pholipids in rat brain chromatin preparations after the in vivoaction of cisplatin. It is impossible to exclude the significanceof chromatin lipids quantitative alterations for cisplatin anti-tumor effects realization. Unfortunately the using of thisdrug is greatly limited because of its severe side effects. Theresults of recently investigations established that combin-ation of steroid hormone estradiol and cisplatin in chemo-therapy treatment schemes decreased cisplatin inducedtoxicities (Ghasemi et al., 2016).

Taking intj foregoing consideration, it may be interest-ing to explore the cisplatin and estradiol joint in vivoaction on total quantities of rat brain chromatin phospholi-pids as well as changes of their individual frac-tions content.

Our results revealed reliably decrease (by 24%) of thetotal amount of phospholipids from rat brain chromatinpreparations after treatment with the antitumor drug cis-platin whereas the combinated injection of cisplatin andestradiol restored the baseline level. Joint use of cisplatinand estradiol lead to recovery the compliance rate ofphosphatidylcholine and phosphatidylethanolamine content.Unlike this the quantity of sphingomyelin and phosphati-dylinositol was increased in comparison to baseline as wellas cisplatin treatment variants. Cisplatin and estradiol com-binated treatment provides significant effect on cardiolipincontent. The absolute quantity of this phospholipidreduced by 38% instead of 15% in case of cisplatinalone treatment.

Changes in content of different phospholipids fractionscaused by cisplatin in vivo action may play a definite role innuclear lipid metabolic pathways. These changes should beconsidered particular negative side effects during the basicantitumor effect of cisplatin. However, the changes of phos-pholipids in case of cisplatin and estradiol combinated treat-ment may be helpful for reducing of cisplatin toxicity andeliminating of its side effects.

References

Ghasemi, M., Nematbakhsh, M., Pezeshki, Z., Soltani, N., Moeini, M., &Talebi, A. (2016). Nephroprotective effect of estrogen and progester-one combination on cisplatin-induced nephrotoxicity in ovariectom-ized female rats. Indian Journal of Nephrology, 26(3), 167–175.

Kuvichkin, V. V. (2016). DNA-Lipids-Me2þ complexes structure and theirpossible functions in a cell. Journal of Chemical Biology andTherapeutics, 1(1), 1–6.

42. Click chemistry in silico andmolecular simulation studies toidentify novel HIV-1 entry inhibitorscaffolds targeting CD4-binding siteof the envelope gp120 protein

Alexander M. Andrianova, Grigory I. Nikolaevb, Yuri V.Kornoushenkoa and Alexander V. Tuzikovb


aInstitute of Bioorganic Chemistry, National Academy of Sciencesof Belarus, Minsk 220141, Belarus; bUnited Institute of InformaticsProblems, National Academy of Sciences of Belarus, Minsk 220012,Belarus [email protected]

In silico design of novel potential HIV-1 entry inhibitors ableto block CD4-binding site of the envelope gp120 proteinwas carried out based on the click chemistry concept. Indoing so, a Drug-Like subset of the ZINC database (Irwin,Sterling, Mysinger, Bolstad, & Coleman, 2012) was screenedby the DataWarrior program to generate two virtualmolecular libraries. Library 1 comprised small moleculeswith an azide group or an alkyne group and aromatic frag-ments critically important for the HIV-1 attachment to cellu-lar receptor CD4. In library 2, all low-molecular compoundswith an azide group or an alkyne group were collected.The modular units from libraries 1 and 2 were then used asreagents to mimic the click chemistry reaction of azide-alkyne cycloaddition by the AutoClickChem program(Durrant & McCammon, 2012). This resulted in a set of 1655 301 hybrid molecules. 294 378 compounds that fullysatisfied Lipinski’s ‘rule of five’ (Lipinski, Lombardo, Dominy,& Feeney, 2001) were further screened by high-throughputdocking and molecular dynamics simulations to evaluatethe affinity of their binding to the target protein. As aresult, five top hits (Figure 1) that exhibited strong attach-ment to the two well-conserved hotspots of the gp120CD4-binding site were selected for the final analysis. In ana-logy to CD4, the identified compounds form hydrogenbonds with Asp-368gp120 and multiple van der Waals con-tacts with the gp120 residues that bind to Phe-43CD4,resulting in destruction of the critical interactions of gp120with Phe-43CD4 and Arg-59CD4. The complexes of the CD4-mimetic candidates with gp120 show relative conform-ational stability within the molecular dynamics simulationsand expose the high percentage occupancies of intermo-lecular H-bonds, in line with the low values of binding freeenergy. In this context, the identified compounds are con-sidered as good scaffolds for the development of new func-tional antagonists of viral entry with broad HIV-1neutralization.

References

Durrant, J. D., & McCammon, J. A. (2012). AutoClickChem: Click chemistryin silico. PLoS Computational Biology, 8(3), e1002397

Irwin, J. J., Sterling, T., Mysinger, M. M., Bolstad, E. S., & Coleman, R. G.(2012). ZINC: A free tool to discover chemistry for biology. Journal ofChemical Information and Modeling, 52(7), 1757–1768.

Lipinski, C. A., Lombardo, F., Dominy, B. W., & Feeney, P. J. (2001).Experimental and computational approaches to estimate solubilityand permeability in drug discovery and development settings.Advanced Drug Delivery Reviews, 46(1-3), 3–26.

43. Computational design of novel1,2,4-triazole-based compounds aspotential aromatase inhibitors

Alexander M. Andrianova, Grigory I. Nikolaevb, Yuri V.Kornoushenkoa and Sergei A. UsanovaaInstitute of Bioorganic Chemistry, National Academy of Sciencesof Belarus, Minsk 220141, Belarus; bUnited Institute of InformaticsProblems, National Academy of Sciences of Belarus, Minsk 220012,Belarus [email protected]

In women organism during the fertile phase, estrogen syn-thesis occurs mainly in the ovaries. However, the intensity ofestrogen synthesis in the ovaries decreases in postmeno-pause associated with about a third of cases of breast can-cer. At this phase, estrogens synthesized in the peripheraltissues using the cytochrome P450 enzyme complex, calledaromatase. This complex consists of the heme-containingcytochrome P450 (CYP19A1) protein and flavoproteinNADPH-cytochrome P450 reductase. Aromatase that isencoded by a single large gene, CYP19A1, catalyzes conver-sion of androgens to estrogens and exhibits biological activ-ity in both peripheral target tissues and in the mammarytumor tissues, providing a high level of estrogen concentra-tion. In estrogen-dependent malignant neoplasms, estrogensact as growth factors for tumor development. Therefore,inhibition of aromatase results in a decrease in the level ofestrogen in the organism and prevention of the growth andspread of cancer cells.

The third-generation aromatase inhibitors (AIs), which arenow used as first-line therapy in the treatment of early- andadvanced-stage breast cancer in postmenopausal women,include two categories: the reversible nonsteroidal inhibitorsanastrozole and letrozole and the steroidal inhibitor exemes-tane. Although these AIs are currently popular and effective

Figure 1. Chemical structures of the potential HIV-1 entry inhibitors. The atoms of hydrogen, oxygen and nitrogen forming hydrogen bonds in the dynamic struc-tures of the complexes between the ligands and HIV-1 gp120 protein are numbered.


in the treatment of postmenopausal estrogen receptorpositive breast cancer, the search for novel drugs stillremains necessary to avoid the risk of possible emergingresistances to available drugs as well as to reduce toxicityand undesirable side effects associated with a pro-longed use.

In this study, computational development of novel tri-azole-based AIs was carried out followed by evaluation oftheir antitumor activity by tools of molecular modeling. Indoing so, in silico design of potential AIs candidates fullysatisfying the Lipinski’s ‘rule of five’ was performed usingthe concept of click chemistry. Complexes of these drug-like molecules with the enzyme were then simulated bymolecular docking and optimized by semiempirical quan-tum chemical method PM7. To identify the most promisingcompounds, stability of the PM7-based ligand/aromatasestructures was estimated in terms of the values of bindingfree energy and dissociation constant. As a result, eighthits that specifically interact with the aromatase catalyticsite and exhibit the high-affinity ligand binding wereselected for the final analysis. All these compounds areshown to coordinate the iron atom of the aromataseheme group and form multiple van der Waals contactswith the critically important residues of the enzyme hydro-phobic pocket, such as Arg-115, Ile-133, Phe-134, Trp-224,!hr-310, Val-370, Met-374, Leu-477 and Ser-478. Six ofeight compounds form hydrogen bond with Met-374 mim-icking the interaction of aromatase with the natural sub-strate androstenedione. In addition, individual ligands arealso involved in specific p- or T-stacking interactions withthe pyrrole rings of the enzyme heme group as well asparticipate in hydrogen bonding with Thr-310, Leu-372,Leu-477 and Ser-478. The selected AIs candidates showstrong attachment to the enzyme active site, in line withthe low values of binding free energy and dissoci-ation constant.

Taken together, the data obtained suggest that the identi-fied compounds may present good scaffolds for the develop-ment of novel potent drugs against breast cancer. Surely, theproperties of these virtual compounds warrant further bio-logical characterization as cellular assays to confirm in vitrotheir interesting in silico profile.

44. Computational modeling andengineering of allosteric regulatorymechanisms in signaling proteins:integration of multiscale simulations,network biology andmachine learning

Gennady M. Verkhivkera,b, Steve Agajaniana,Nathaniel Bischoffa, Lindy Astla and KristinBlacklocka,caDepartment of Biomedical and Pharmaceutical Sciences, SchmidCollege of Science & Technology and School of Pharmacy,Chapman University, Irvine, CA 92866, USA; bDepartment ofPharmacology, University of California San Diego, San Diego, CA92093, USA; cDepartment of Chemistry and Chemical Biology,Rutgers University, Piscataway, NJ 08854, USA

[email protected]

The allosteric interactions allow for molecular communicationand event coupling in signal transduction pathways and net-works. The overarching goal of understanding molecularprinciples underlying recognition of protein kinase clientsand chaperone-based modulation of kinase activity is funda-mental to activity of many signaling proteins. The synergisticroles of the Hsp90-Cdc37 chaperone machinery and proteinkinases in biology and disease have stimulated extensivestructural and functional studies of regulatory mechanisms.Allosteric interactions of the Hsp90 with cochaperones andprotein kinase clients (left panel of Figure 1) derived fromrecent crystal structures (Verba & Agard, 2017) can determineregulatory mechanisms and cellular functions of many signal-ing proteins and cascades. We report the results of multiscalesimulations and network biology studies of the Hsp90 chap-erones and a panel of protein kinases with an atomic-levelanalysis of regulatory dynamic changes and binding of theseprotein systems in signaling networks (Czemeres, Buse, &Verkhivker, 2017). The results of biophysical and computa-tional biology studies combined with biochemical experi-ments have been used to derive a network model of kinaseregulation by Hsp90 chaperones (shown on the right panel

Figure 1.


of Figure 1). Among our primary findings is the evidencethat a small number of functional motifs may be utilized bythe chaperone and protein kinases to act as central regula-tors of protein client binding in signaling networks. Thiswork has offered atomistic insights into mechanisms of kin-ase activation by molecular chaperones orchestrated by dir-ection-specific cross-talk between key regulatory regions. Thedeveloped platform of atomistic simulations and networkbiology approaches is also integrated with machine learning-based models of allosteric regulation for oncogenic proteinsin signaling cascades.

Funding

This research has been supported by institutional fundingfrom Chapman University.

References

Czemeres, J., Buse, K., & Verkhivker, G. M. (2017). Atomistic simulationsand network-based modeling of the Hsp90-Cdc37 chaperone bindingwith Cdk4 client protein: A mechanism of chaperoning kinase clientsby exploiting weak spots of intrinsically dynamic kinase domains.PLoS One, 12(12), e0190267.

Verba, K. A., & Agard, D. A. (2017). How Hsp90 and Cdc37 lubricate kin-ase molecular switches. Trends in Biochemical Sciences, 42(10),799–811.

45. Constraining evolution—avoidingdrug resistance: lessons from viruses

Celia SchifferInstitute for Drug Resistance, University of Massachusetts MedicalSchool, Worcester, MA 01655, USA

Drug resistance negatively impacts the lives of millions ofpatients and costs our society billions of dollars by limitingthe longevity of many of our most potent drugs. Drugresistance can be caused by a change in the balance ofmolecular recognition events that selectively weakensinhibitor binding but maintains the biological function ofthe target. To reduce the likelihood of drug resistance, adetailed understanding of the target’s functionis necessary.

Both structure at atomic resolution and evolutionarily con-straints on its variation is required. This rationale was derivedfrom our lab’s experience with substrate recognition anddrug resistance in HIV, HCV and Influenza. In particular, wehave acquired a rich and versatile experimental dataset ofviral proteases altered by the selective pressures of inhibitors.With these data, we are integrating alterations in both the pro-tein sequence and the inhibitor with changes in potency andwe correlate this data to our co-crystal structures and our strat-egy of parallel molecular dynamics (pMD) to elucidate molecu-lar mechanisms of drug resistance.

pMD is a strategy we have developed to collectively ana-lyze a series of MD simulations of similar yet distinct molecu-lar complexes to decipher conformational and dynamic

differences responsible for changes in molecular recogni-tion. We perform pMD simulations on complexes withvarying protein sequence and ligand identity to unravelstructural and dynamic properties that underlie coupledchanges in molecular recognition and resistance. We haveapplied this pMD strategy to protein-ligand complexes forseries of natural substrates, inhibitors and protease muta-tions. Combined with experimental potency data, we deter-mine which physical properties or molecular interactions,including with water, are key to molecular recognition fora given system.

These changes in structure and dynamics dictate theinterdependency of molecular mechanism of resistanceand are principals that are generally applicable to otherquickly evolving diseases where drug resistance isquickly evolving.

References

Leidner, F., Kurt Yilmaz, N., Paulsen, J., Muller, Y. A., & Schiffer, C. A.(2018). Hydration structure and dynamics of inhibitor-bound HIV-1protease. Journal of Chemical Theory and Computation, 14(5),2784–2796. doi: 10.1021/acs.jctc.8b00097. Epub 2018 Apr 18.

Matthew, A. N., Leidner, F., Newton, A., Petropoulos, C. J., Huang, W., Ali,A., … Schiffer, C. A. (2018). Molecular mechanism of resistance in aclinically significant double-mutant variant of HCV NS3/4A protease.Structure, 26(10), 1360–1372.e5. doi: 10.1016/j.str.2018.07.004. Epub2018 Aug 23.

€Ozen, A., Lin, K. H., Kurt Yilmaz, N., & Schiffer, C. A. (2014). Structuralbasis and distal effects of Gag substrate coevolution in drug resist-ance to HIV-1 protease. Proceedings of the National Academy ofSciences of the United States of America, 111(45), 15993–15998. doi:10.1073/pnas.1414063111. Epub 2014 Oct 29.

Paulsen, J. L., Leidner, F., Ragland, D. A., Kurt Yilmaz, N., & Schiffer, C. A.(2017). Interdependence of inhibitor recognition in HIV-1 protease.Journal of Chemical Theory and Computation, May 913(5), 2300–2309.doi: 10.1021/acs.jctc.6b01262. Epub 2017 Apr 11.

Prachanronarong, K. L., €Ozen, A., Thayer, K. M., Yilmaz, L. S., Zeldovich,K. B., Bolon, D. N., … Schiffer, C. A. (2016). Molecular basis for differ-ential patterns of drug resistance in influenza N1 and N2 neuraminid-ase. Journal of Chemical Theory and Computation, 12(12), 6098–6108.Epub 2016 Nov 17.

Ragland, D. A., Nalivaika, E. A., Nalam, M. N., Prachanronarong, K. L., Cao,H., Bandaranayake, R. M., … Schiffer, C. A. (2014). Drug resistanceconferred by mutations outside the active site through alterations inthe dynamic and structural ensemble of HIV-1 protease. Journal of theAmerican Chemical Society, 136(34), 11956–11963. doi: 10.1021/ja504096m. Epub 2014 Aug 18.

Soumana, D. I., Kurt Yilmaz, N., Ali, A., Prachanronarong, K. L., & Schiffer,C. A. (2016). Molecular and dynamic mechanism underlying drugresistance in genotype 3 hepatitis C NS3/4A protease. Journal of theAmerican Chemical Society, 138(36), 11850–11859. doi: 10.1021/jacs.6b06454. Epub 2016 Sep 2.

46. De novo aggregationof alzheimer’s Ab25-35 peptidesin a lipid bilayer

Amy K. Smith and Dmitri K. KlimovSchool of Systems Biology, George Mason University, Manassas,VA 20110, USA [email protected]


One of the mechanisms of cytotoxicity attributed toAlzheimer’s Ab peptides postulates that their aggregationdisrupts membrane structure causing uncontrollable perme-ation of Ca2þ ions. To gain molecular insights into theseprocesses, we have performed all-atom explicit solvent rep-lica exchange with solute tempering (REST) moleculardynamics simulations probing aggregation of the naturallyoccurring Ab fragment Ab25-35 within the DMPC lipidbilayer. To compare the impact produced on the lipid bilayerby Ab25-35 oligomers and monomers, we used as a controlour previous simulations, which explored binding of Ab25-35monomers to the same bilayer. We found that compared tomonomeric species aggregation results in much deeperinsertion of Ab25-35 peptides in the hydrophobic core of thelipid bilayer causing more pronounced disruption in its struc-ture. Our study indicates that Ab25-35 peptides aggregateby incorporating monomer-like structures with stable C-ter-minal helix. As a result, the Ab25-35 dimer features unusualhelix head-to-tail topology supported by a parallel off-regis-try interface. The head-to-tail topology readily affords furthergrowth of an aggregate by recruiting additional peptides.Free-energy landscape of Ab25-35 binding and aggregationreveals that inserted dimers represent the dominant equilib-rium state augmented by two metastable states associatedwith surface bound dimers and inserted monomers. Analysisof the free-energy landscape allows us to propose the path-way and mechanism of Ab25-35 binding, aggregation andinsertion into the lipid bilayer. Our all-atom explicit solventsimulations provide the first, to our knowledge, all-atomdescription of equilibrium de novo Ab peptide aggregationmediated by a lipid bilayer.

47. Dopamine channels withapplications

Zhengqiong Zhang, Xiaowen Zhou, Zeren Xu,Chengqi Lu and Sichuan Xu

College of Chemical Science and Technology and Pharmacy,Yunnan University, Kunming, 650091, P. R. China

[email protected]

Dopamine molecules, by combining different sub-types ofdopamine receptors, regulate the motor function, cognitiveactivity, and emotions of living organisms, which are closelyrelated to the pathology and treatment of Parkinson’s dis-ease and schizophrenia (Ye et al., 2013; Li et al., 2017; Xieet al., 2017). In vivo, the production of dopamine moleculesis by midbrain dopaminergic neuronal tissue. In the substan-tia nigra tissue, dopamine molecules are produced in thedopaminergic neuron cells of the anterior membrane, andthe produced dopamine molecules packaged by the vesiclesare released into the intercellular space through the cellmembrane. At the intercellular space, various dopaminereceptors are present on the dopaminergic neuron cell mem-branes of the posterior membrane cells. These receptors canaccept the binding by dopamine molecules to exert dopa-mine function. However, dopamine molecules placed in theintercellular space had long been not cared. No one hadstudied the actual part of the dopamine receptors for dopa-mine molecules to act. No one had studied for dopaminemolecules how to work actually. And no one had studied thepresence of dopamine molecular pathways throughout thedopamine receptors. We have used the dopamine thirdreceptor crystal structure (Chien et al., 2010), to study thecomplex structure of the dopamine molecule with thirdreceptor protein, to study and obtain the dopamine activepocket that exert the functions (Jin et al., 2011), and to studyand obtain dopamine molecular channels within the dopa-mine receptor structures (Li et al., 2017).

There are two types of dopamine molecular channelswithin the dopamine receptor to be found by us, one is‘dopamine functional channel’ and the other is ‘dopamineprotective channel’. Then, we have further proved theexistence of ‘dopamine functional channel’ and the exist-ence of ‘dopamine protective channel’ by a combinationof molecular dynamics experiments and animalexperiments.

Due to the presence of dopamine functional channel, thedopamine molecules released into the intercellular space canbe divided into two categories. A class of dopamine mole-cules that can reach the functional sites through the dopa-mine functional channel can truly function as a signaltransduction and regulation. These functional dopamine mol-ecules are called ‘functional dopamine’. In addition, anotherclass of dopamine molecules, which are nonfunctional dopa-mine molecules, will be reabsorbed or decomposed.Obviously, functional dopamine occupies a small fraction oftotal dopamine, similar to drinking water occupying a smallfraction of total water. The relationship between functionaldopamine content and total dopamine content, we havestudied to establish a formula to represent:

GD ¼ f ( TD (1)

wherein GD is a functional dopamine content; TD is the totaldopamine content (expressed as a percentage); f is the


dopamine functionalization factor, which is the ratio of totaldopamine converted to functional dopamine, normalized to1 as a value for normal healthy persons.

Based on the above formula (1), we have clarified theessence of Parkinson’s disease to be the lack of functionaldopamine, and proposed the scientific pathology ofParkinson’s disease that is a significant reduction in func-tional dopamine to cause Parkinson’s disease. We can scien-tifically name Parkinson’s disease as ‘Functional dopaminedeficiency syndrome’. According to the scientific pathologyof Parkinson’s disease, all types of Parkinson’s disease can beincluded and confirmed without misdiagnosis and misseddiagnosis. According to the scientific pathology ofParkinson’s disease, new drugs for treating Parkinson’s dis-ease can be found. Depending on the types of Parkinson’sdisease, different therapy programs and new drugs can beused able to cure the disease of patients with early and mid-stage Parkinson’s disease, and feasibly better to controlpatients with the late advanced Parkinson’s disease for keep-ing better symptoms. Our research can make Parkinson’s dis-ease from a difficult, complex, incurable and uncontrollabledisabling disease changed to be a simple, curable or control-lable disease, although it cannot be eradicated at present.

Funding

Support from the National Natural Science Foundation ofChina (21163024, 21563032).

References

Chien, E. Y., Liu, W., Zhao, Q., Katritch, V., Han, G. W., Hanson, M. A., Shi,L., & Stevens, R. C. (2010). Structure of the human dopamine D3receptor in complex with a D2/D3 selective antagonist. Science,330(6007), 1091–1095.

Jin, Y., Wang, Y., Bian, F. Y., Shi, Q., Ge, M. F., Wang, S., … Xu, S. C.(2011). Three-dimensional structure of dopamine 3-subtype receptorwith the active site residues for the binding of dopamine. ActaPhysico-Chimica Sinica, 27, 2432–2446.

Li, A., Xie, W., Wang, M., & Xu, S. C. (2017). Molecular dynamics of dopa-mine to transmit through molecular channels within D3R. ActaPhysico-Chimica Sinica, 33, 927–940.

Xie, W., Wang, M., Li, A., & Xu, S. C. (2017). Molecular dynamics simula-tion of d-benzedrine transmitting through molecular channels withinD3R. Journal of Biomolecular Structure and Dynamics, 35(8),1672–1684.

Ye, N., Neumeyer, J. L., Baldessarini, R. J., Zhen, X. C., & Zhang, A. (2013).Recent progress in development of dopamine receptor subtype-selective agents: Potential therapeutics for neurological and psychi-atric disorders. Chemical Reviews, 113(5), PR123–PR178.

48. Elucidating the molecularinteraction of noscapine with bovineserum albumin (BSA): spectroscopicand molecular dynamics approach

Chhaya Chaudhary, Damini Sood andRamesh ChandraDepartment of Chemistry, University of Delhi, Delhi 110007, India

[email protected]

The interaction of the drug with serum albumin forms anintegral part of the pharmacokinetic profile of the drug. Thepresent study employed various spectroscopic techniquesi.e., fluorescence, UV-Visible and circular dichroism andmolecular dynamics approach to investigate the mechanismof interaction of an anticancer drug, Noscapine (Ns) withBovine Serum Albumin (BSA). Ns binding to BSA leads to the

Figure 1.


quenching of intrinsic fluorescence of BSA, and the mechan-ism was a static one. The CD spectra showed that Ns bindingto BSA leads to a loss of alpha helicity of the protein. The FT-IRspectra further confirmed the changes in secondary structureof BSA upon interacting with Ns. The in vitro results were vali-dated with molecular docking and dynamics techniques.Molecular docking showed strong interactions with a highscore. Molecular dynamics simulation analysis also suggestedthe stable binding with lower deviation in RMSD and RMSFvalues through persistent long simulation run. The findingsthus obtained were in good relation with the spectroscopicdata. These results suggest the optimal diffusion of Ns into thebloodstream for the treatment of cancer (Figure 1).

References

Checchi, P. M., Nettles, J. H., Zhou, J., Snyder, J. P., & Joshi, H. C. (2003).Microtubule-interacting drugs for cancer treatment. Trends inPharmacological Sciences, 24(7), 361–365.

Lu, Y., Chen, J., Xiao, M., Li, W., & Miller, D. D. (2012). An overview oftubulin inhibitors that interact with the colchicine binding site.Pharmaceutical Research, 29(11), 2943–2971.

Naik, P. K., Lopus, M., Aneja, R., Vangapandu, S. N., & Joshi, H. C. (2012).In silico inspired design and synthesis of a novel tubulin-binding anti-cancer drug: Folate conjugated noscapine (Targetin). Journal ofComputer-Aided Molecular Design, 26(2), 233–247.

49. Entrapment of flow of substrateto diversion pathway: strategyto combat persistent tuberculosis

Indira GhoshSchool of Computational & Integrative Sciences, Jawaharlal NehruUniversity, New Delhi, India

The post genomic era has provided the leap into the quantita-tive biology and introduced biologists into the ‘omes’ and ‘ics’world. For biologists, it is utmost important to know the biol-ogy of disease and the mechanism of action of medicines. Butno longer are these contained in the domain of physiologyand medicinal chemistry. The importance of reorganizing ourthinking process is mostly reflected in the merger and acquisi-tion of Biotechnology, Bioinformatics and Chemoinformaticscompanies by/with large pharmaceutical companies recently.

The development in the field of molecular modeling andsimulation has played the leading role in the drug designtechnology in pharmaceutical industry. Starting fromGenomics, alignment of genes of interest amongst the spe-cies, three-dimensional structure prediction from the know-ledge of sequence of amino acids, prediction of active sites,searching for lead compounds using database and structure-based design, prediction of binding efficiency prior to theexperiment and lead optimization using physico-chemicalproperties of the compounds are a few to cite. One suchcase study using in silico attempt towards the designingfrom known antituberculosis chemicals a set of novel inhibi-tor and finding the drug target(s) using pathway simulationand analysis (Prakash & Ghosh, 2006; Singh & Ghosh, 2006;Vinekar & Ghosh, 2009) will be discussed in brief.

Recent news has warned the medical community that waragainst pathogenic bacteria is yet not over, repeated breakingof different drug resistance in different ranges of pathogen hasbeen observed in the patient, especially in developing andunderdeveloped countries. Many researchers, probably thenext to cancer are experimentally using many prong attack onthe understanding the mechanism and developing new strat-egy of inhibitors for combating this phenomena, resurgingXDR, MDR, PDR. The extensive discussion to define theseclasses is available in the literature (Diacon, 2014; Iseman,1993; K€oser et al. 2015; Magiorakos, 2011). Research workengaging with clinician, epidemiologist, biologists, microbiolo-gist, etc. in this field is emerging to highlight the complexity ofthe problem. However, many chemicals have been developedto combat the MDR/XDR Tb yet not been fully successful for along time. Tuberculii, bacteria have shown to have multiplespontaneous mutations at a predictable rate and independentof different drug administrations. Such phenomena are preva-lent to many bacterial and more common to viral diseasewhich leads researchers to develop multiple targetdrug designing.

Present discussion will open up the future of understand-ing of disease, its inter relationship within biochemical path-way and effect of protein–protein interaction identified astarget for combating persistence stage in bacteria.

References

Diacon, A. H. (2014). Multidrug-resistant tuberculosis and culture conversionwith bedaquiline. New England Journal of Medicine, 371, 8. nejm.org

Iseman, M. D. (1993). Treatment of multidrug-resistant tuberculosis. NewEngland Journal of Medicine, 329, 784–791.

K€oser, C. U., Javid, B., Liddell, K., Ellington, M., Feuerriegel, J., Niemann, S., …T€or€ok, M. E., et al. (2015). Drug-resistance mechanisms and tuberculosisdrugs. Lancet, 385(9965), 305–307. doi:10.1016/S0140-6736(14)62450-8.

Magiorakos, A.-P. (2011). Multidrug-resistant, extensively drug-resistantand pan drug-resistant bacteria: An international expert proposal forinterim standard definitions for acquired resistance. ClinicalMicrobiology and Infection, doi:10.1111/j.1469-0691.2011.03570.x

Prakash, O., & Ghosh, I. (2006). Developing an antituberculosis com-pounds database & data mining in the search of a motif responsiblefor the activity of a diverse class of Antituberculosis agents. Journal ofChemical Information and Modeling, 46, 17–23.

Singh, V. K., & Ghosh, I. (2006). Kinetic modeling of tricarboxylic acidcycle and glyoxylate bypass in Mycobacterium tuberculosis, and itsapplication to assessment of drug targets. Theoretical Biology andMedical Modelling, BMC Journal, 3(3), 27.

Vinekar, R., & Ghosh, I. (2009). Determination of phosphorylation sites forNADP-specific isocitratedehydrogenase from Mycobacterium tubercu-losis. Journal of Biomolecular Structure and Dynamics, 26(6), 663–895.

50. Identification of potent and novelphosphatidylinositol 3-Kinase catalyticsubunit alpha inhibitors: a structure-based drug design approach

Geet Madhukar and Naidu SubbaraoSchool of Computational and Integrative Sciences, JawaharlalNehru University, New Delhi, India



Head and neck squamous cell carcinoma (HNSCC), one ofthe most common causes of deaths due to cancers in AsianCountries and the sixth most common cause of cancer glo-bally is a heterogeneous group of upper aerodigestive tractmalignancies (Saleh et al., 2018). Phosphatidylinositol 3-kin-ase catalytic subunit alpha (PIK3CA) is reported to be com-monly altered in many human tumors including HNSCC(Wen & Grandis, 2015). Phosphatidylinositol-3-kinases (PI3Ks)are lipid kinases involved in the regulation of cell survival,growth and metabolism. PI3 Kinase phosphorylates PI(3,4)P2(PIP2) converting it to PI(3,4,5)P3 (PIP3). Alterations such asmutation, gene amplification and overexpression of PIK3CA arecommonly reported in HNSCC (Garc"ıa-Escudero et al., 2018).This leads to irregulated cell growth due to improper activationof p110a enzymatic activity. The present study aims to find outthe potent and novel inhibitors of PIK3CA isoform of PI3Ks.Extensive database screening from NCI, Life Chemical (KinaseFocused Library Similarity search, Kinase Screening Library, LCKinase docking) and ChEMBL (KinaseSARfari) were performed.Computational approaches were employed to carry out aStructure-Based Drug Design study. Screened compounds fromNCI and KinaseSARfari showed superior results and surpassedthe benchmark compounds with huge differences. Top com-pounds of NCI (NSC 671560, NSC 729878) and KinaseSARfari(ChEMBL43781) reported GLIDE Gscore of % 15.73 and% 17.55 kcal/mol, respectively whereas benchmark compoundsfrom ChEMBL43781 had maximum GLIDE Gscore of% 10.36 kcal/mol. Also, NVP-BYL719 (PDB:4JPS) the only reportedselective inhibitor of PIK3CA has GLIDE Gscore % 8.16 kcal/mol.To validate our results the top ranking compounds were dockedusing GOLD and the results were consistent. Along with GLIDEand GOLD scores, the binding affinity of the protein-ligand com-plex was also scored using X-Score program and found consist-ent with the previous results. A total of 10 compounds from allthe screened libraries were selected and are currently under in-vitro investigation, for further validation of our findings.

References

Garc"ıa-Escudero, R., Segrelles, C., Due~nas, M., Pombo, M., Ballest"ın, C.,Alonso-Ria~no, M., … Lorz, C. (2018). Overexpression of PIK3CA inhead and neck squamous cell carcinoma is associated with poor out-come and activation of the YAP pathway. Oral Oncology, 79, 55–63.

Saleh, K., Eid, R., Haddad, F. G. H., Khalife-Saleh, N., & Kourie, H. R. (2018).New developments in the management of head and neck cancer—impact of pembrolizumab. Therapeutics and Clinical Risk Management,14, 295–303.

Wen, Y., & Grandis, J. R. (2015). Emerging drugs for head and neck can-cer. Expert Opinion on Emerging Drugs, 20(2), 313–329.

51. Identification of novel ureaseinhibitors: pharmacophore modeling,virtual screening and moleculardocking studies

Richa Arora, Upasana Issar and Rita KakkarComputational Chemistry Laboratory, Department of Chemistry,University of Delhi, Delhi 110 007, India

[email protected]

Pharmacophore modeling and atom based 3D-QSAR havebeen developed on N-acylglycino- and hippurohydroxamicacid derivatives, which are known potential inhibitors of ure-ase. This is followed by virtual screening and ADMET (absorp-tion, metabolism, excretion and toxicity) studies on a largelibrary of known drugs in order to get lead molecules asHelicobacter pylori urease inhibitors. A suitable three-featuredpharmacophore model comprising one H-bond acceptor andtwo H-bond donor features (ADD.10) has been found to bethe best QSAR model (Figure 1). An external library of com-pounds (" 3000 molecules), prefiltered using Lipinski’s ruleof five, has been further screened using the pharmacophoremodel ADD.10. By analyzing the fitness of the hits withrespect to the pharmacophore model and their bindinginteraction inside the urease active site, four molecules havebeen identified as extremely good urease inhibitors. Two ofthese have significant potential and should be taken up forfurther drug designing process.

52. Identification of potentialinhibitors for AroG againstMycobacterium tuberculosis

Nalamolu Ravina Madhulitha, Katari Sudheer Kumar,Chiranjeevi Pasala, Sivaranjani Pakala and AmineniUmamaheswariBioinformatics Centre, Department of Bioinformatics, SriVenkateswara Institute of Medical Sciences University, Tirupati517507, Andhra Pradesh, India [email protected]

Infections by opportunistic bacteria have significant contribu-tions to morbidity and mortality in humans. Tuberculosis hasmany manifestations affecting bone, central nervous systemand many other organ systems. Tuberculosis is primarily apulmonary disease that is initiated by the deposition ofMycobacterium tuberculosis (M. tb) contained in aerosol drop-lets onto lung alveolar surfaces. Developing potent inhibitors

Figure 1. Proposed pharmacophore model.


is an important strategy to tackle the pathogenecity of M. tbstrains. Fourteen crystal structures of Phospho-2-dehydro-3-deoxyheptonate aldolase (AroG) are available in the proteindata bank (PDB). AroG of M. tb possess 3 co-crystal structures(2B7O, 3NV8 and 3PFP) with substrate (PEP) and inhibitor(O35) in the PDB. Among the two diverse co-crystal struc-tures (3NV8 and 3PFP), the best resolution structure (3NV8)was considered for structure based drug design to proposeantagonists through swiss similarity screening, dockings andmolecular dynamics simulations. O35 and PEP of AroG werescreened against swiss similarity search of 31 databases com-prising 31,11,77,426 compounds obtained 2858 structuralhits. The hits were processed through rigid receptor docking(RRD), quantum polarized ligand docking (QPLD) and bindingfree energies were calculated by Prime-MM/GBSA approach(Madhulitha et al., 2017) with AroG. RRD followed by MM-GBSA calculations to the generated library of AroG resulted29 compounds, upon comparison with crystal ligands 11compounds were scored better. To define the leads, 11 com-pounds were subjected to QPLD and binding free energy cal-culations revealed 5-leads on comparison with PEP and O35,lead1 possess the least binding free energy and better bind-ing affinity. Lead1 and PEP have QPLD XP Gscore of % 8.490and % 6.487 kcal/mol, DGscore of % 51.80 and % 35.209 kcal/mol, respectively. The stability of AroG-lead1 complex wasbetter than AroG-PEP complex in natural physiological condi-tions using Desmond v5.3 for 100 ns molecular dynamicssimulations (Katari et al., 2016; Pasala et al., 2018) revealedthe least potential energy (% 179483.298 kcal/mol), proteinbackbone RMSD (3.1183Å), protein backbone RMSF(1.1317Å), lead1 RMSD (2.8421Å), lead1 RMSF (0.9607Å) with15,031 protein-ligand contacts. The proposed lead1 possessfavorable ADME/T properties and acts as the best AroGantagonist of M. tb (Figure 1).

Acknowledgments

NRM is highly acknowledged to ICMR for sanctioning SRF (ISRM/11(21)/2017). Authors are thankful to DBT, Ministry of Science and Technology,Government of India, New Delhi for supporting the work throughBTISnet BIF program (No. BT/BI/25/037/2012).

References

Katari, S. K., Natarajan, P., Swargam, S., Kanipakam, H., Pasala, C., &Umamaheswari, A. (2016). Inhibitor design against JNK1 through e-pharmacophore modeling docking and molecular dynamics simula-tions. Journal of Receptor and Signal Transduction Research, 36(6),558–571.

Madhulitha, N. R., Pradeep, N., Sandeep, S., Hema, K., & Chiranjeevi, P.(2017). E-Pharmacophore model assisted discovery of novel antago-nists of nNOS. Biochemistry & Analytical Biochemistry, 6, 307.doi:10.4172/2161-1009.1000307

Pasala, C., Chilamakuri, C. S. R., Katari, S. K., Nalamolu, R. M., Bitla, A. R., &Umamaheswari, A. (2018). Epitope-driven common subunit vaccinedesign against H. pylori strains. Journal of Biomolecular Structure andDynamics. doi: 10.1080/07391102.2018.1526714.

53. In silico assessment ofprogesterone receptor ligand bindingdomain using molecular dynamic:a new insight into breastcancer treatment

Vahid Zarezadea, Marzie Abolghasemia, FakherRahimb,c and Ali VeisiaaBehbahan University of Medical Sciences, Iran; bHealth ResearchInstitute, Thalassemia and Hemoglobinopathies Research Centre,Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran;cMetabolomics and Genomics Research Center, Endocrinology andMetabolism Molecular-Cellular Sciences Institute, Tehran Universityof Medical Sciences, Tehran, Iran [email protected]

Progesterone receptor (PR), also known as a member of thesteroid/nuclear receptor (NR) superfamily, regulates a com-plex network of distinct target genes. Ligand binding to a PRleads to a conformational change in the receptor, which acti-vates PR to bind to DNA and regulates the transcription(Brennan & Lim, 2015). Loss of PR expression is associatedwith worse overall prognosis and survival among patientswith Breast cancer (BC) (Abdel-Hafiz & Horwitz, 2012).Although considerable research has been devoted to BC,rather less attention has been paid to PR analysis. In the fol-lowing research, an extensive in silico analytical study was

Figure 1.


performed to the computational measurement of PR modula-tion by a large number of diverse ligands. In this study, weperformed molecular dynamic (MD) simulation in twophases. In the first phase, the prepared conformation of PRwas used as the initiating structure for MD simulation. Theconstituents of the simulation system were protein andwater. In the 2nd phase, the acquired conformation of PRfrom the first phase of MD besides the docked ligand wasengaged in the MD simulation process. In the dockingresults, we found a hydrogen-bond between this compoundand Arg94 in the PR. Moreover, we docked 12 standarddrugs to the PR, which found best docking g score inLevonorgestrel with highest binding energy (% 8.35 kcal/mol)and lowest ki (0.758 mM). This drug showed interaction withHis31, Asn33, Thr34, Lys35, Pro36, Asp37, Thr38, Ser39, Ser41,Leu42, Asp109, Leu110, Ile111, Leu112 and Arg116 in PR. Toshow the conformational alignment between best dockednatural compound to best docked standard drugs we super-imposed these compounds corresponds to PR (Figure 1).

References

Abdel-Hafiz, H. A., & Horwitz, K. B. (2012). Control of progesterone receptortranscriptional Synergy by sumoylation and desumoylation. BMCMolecular Biology, 13 (Mar 22) article N0. 10: doi:10.1186/1471-2199-13-10

Brennan, M., & Lim, B. (2015). The actual role of receptors as cancermarkers, biochemical and clinical aspects: Receptors in breast cancer.Advances in Experimental Medicine and Biology, 867, 327–337. doi:10.1007/978-94-017-7215-0_20.

54. In silico designing of potentialnoscapine analog andpharmacological evaluation usingchemoinformatics approach

Vartika Tomar, Neeraj Kumar and Ramesh ChandraDrug Discovery & Development Laboratory, Department ofChemistry, University of Delhi, Delhi 110007, India

[email protected]

Noscapine and its derivatives have been explored for itsinteraction with tubulin protein, to be used as strong anti-cancer agent. A detailed computational analysis have beenperformed to determine the noscapine analog to inhibit thetubulin protein with high specificity, overcoming the issuesof non-specificity and side effects associated with potentialinteraction. In this study, noscapine analogs were designed

Figure 1. Predicted binding modes of the top-ranked standard drug to the models of PR. (a) A 2-dimensional form conformation of Levonorgestrel docked to PR.(b) A 3-dimensional form conformation of Levonorgestrel docked to PR. In 3-D format, PR is green and magenta color, while for ligands (Levonorgestrel), carbonatoms are cyan; oxygen atoms are red and nitrogen atoms are blue.

Figure 1.


using the state of art methodology employing alkyl groups,in order to enhance the potency of the drug to bind targettubulin receptor (potential target for cancer treatment).We have performed the molecular docking and moleculardynamics simulation. Also, pharmacological evaluations werealso performed to assess the pharmacokinetic properties ofthe lead compound. Our results illustrated that compound-1possess the strongest interaction with the target tubulin pro-tein with higher binding free energy score of % 8.49 kcal/molamong the whole library and noscapine (% 7.86 kcal/mol).Our results were confirmed by the molecular dynamics simu-lation study of lead compound tubulin complex with lowerRMSD and atomic fluctuation values. Importantly, lead com-pound was found to good pharmacokinetic properties andshowed the potential lead to be used as potential cancertherapeutics (Figure 1).

References

Conde, C., & C"aceres, A. (2009). Microtubule assembly, organization anddynamics in axons and dendrites. Nature Reviews Neuroscience, 10(5),319–332.

Lu, Y., Chen, J., Xiao, M., Li, W., & Miller, D. D. (2012). An overview oftubulin inhibitors that interact with the colchicine binding site.Pharmaceutical Research, 29(11), 2943–2971.

Naik, P. K., Lopus, M., Aneja, R., Vangapandu, S. N., & Joshi, H. C. (2012).In silico inspired design and synthesis of a novel tubulin-binding anti-cancer drug: Folate conjugated noscapine (Targetin). Journal ofComputer-Aided Molecular Design, 26(2), 233–247.

55. In silico identification of leadstargeting interleukin-6 againstpathogenesis of atherosclerosis

Kanipakam Hemaa,d!, Sandeep Swargamb, NatarajanPradeepc and Amineni UmamaheswaridaFunctional Genomics Unit, CSIR-IGIB, New Delhi 110007, India;bInstitute of Molecular Medicine, Jamia Hamdard University, NewDelhi 110062, India; cDepartment of Biotechnology and MedicalEngineering, NIT, Rourkela, Odisha, 769008, India; dBioinformaticsCentre, Department of Bioinformatics, SVIMS University, Tirupati,Andhra Pradesh, 517507, India [email protected]!Presenting author: [email protected]

Atherosclerosis is one of the top, multi-factorial diseasesmajorly responsible for the cause of deaths that makes thelife-threatening clinical events such as acute coronary syn-dromes and stroke. Alongside, the widespread emergence ofbacterial resistance to existing antibiotics has emphasizedthe need to develop novel therapeutics to treat atheroscler-osis with the identification of new targets and novel thera-peutics (Hema, 2015). The common offending organismsinclude Chlamydiophila pneumoniae, Poryphromonas gingiva-lis, Helicobacter pylori, Enterobacter hormaechei, Prevotellanigrescens, Prevotella intermedia, Streptococcus sanguinis,Tannerella forsythia, Aggregatibacter actinomycetemcomitansD11S-1, Aggregatibacter actinomycetemcomitans D7S-1 andEscherichia coli (Hema, 2015). Over expression of human IL-6drives the atherosclerosis disease progression hence,regarded as a prominent target in the present study todesign novel antagonists (Figure 1). Kalai et al., 2014reported that inhibiting the binding affinity of human IL-6and gp-130 effectively blocks the signaling pathway involvedin atherosclerosis progression and also quoted that the resi-dues Ile 29, Ile 32 and Leu 33 Arg 30, Arg 182 are majorlyresponsible for the binding of human IL-6 and gp-130 (Kalaiet al., 1997). The proposed lead 1 had good binding affinity,good docking scores and similar orientation with lead 1were in well conformity with the binding orientation of gp-130 (Figure 2). The additional five hydrogen bonds wereformed with the binding site residues Tyr 31, Leu 33, Glu110, Gln 111, Leu 178 and hydrogen bond with Lys 27 wasobserved in 98% of trajectories, Tyr 31 was observed in 96%and Arg 182 was observed with 79% trajectories strengthenthe stability of lead 1 with human IL-6 with better bindingaffinity. The consistent energy, five water-mediated interac-tions, RMSD and RMSF of the complex was also within thelimit (Figure 3). Hence, the proposed lead can act as poten-tial antagonists for inhibiting the human IL-6 in signalingpathway in the progression of atherosclerosis. The lead mol-ecule would hold as a promising antagonist in drug discov-ery if synthesized and tested in animal models.

Acknowledgments

This research has been supported by DST-INSPIRE, Govt. of India, for theSRF. The authors are thankful to DBT, ministry of science and technol-ogy, Govt. of India for providing all facilities to carry out the work.

Figure 1. Figure 2. Figure 3.


References

Hema, K., Priyadarshini, V., Pradhan, D., Munikumar, M., Sandeep, S.,Pradeep, N., … Umamaheswari, A. (2015). Identification of putativedrug targets and vaccine candidates for pathogens causing athero-sclerosis. Biochemistry & Analytical Biochemistry, 4, 1–9.

Hema, K., Vani Priyadarshini, I., Sandeep, S., Pradeep, N., Chiranjeevi, P., &Umamaheswari, A. (2015). Subunit vaccine design against pathogenscausing atherosclerosis. Journal of Biomolecular Structure andDynamics, 33(suppl. 1)

Kalai, M., Montero-Julian, F. A., Gr€otzinger, J., Fontaine, V.,Vandenbussche, P., Deschuyteneer, R., … Content, J. (1997). Analysisof the human interleukin-6/human interleukin-6 receptor bindinginterface at the amino acid level: Proposed mechanism of interaction.Blood, 89(4), 1319–1333.

56. In silico studies of interaction ofhoechst 33258 within minor grooveof B-DNA

Upasana Issar, Richa Arora and Rita KakkarComputational Chemistry Laboratory, Department of Chemistry,University of Delhi, Delhi 110 007, India

[email protected]

DNA is the most sought after drug design target in theleague of anticancer drugs. Drugs interfere with the replica-tion and transcription of DNA, thereby causing cell death.Hoechst 33258 is a prominent minor groove binder that dis-plays specific binding towards the adenine-thymine (AT) richregion of the minor groove of B-DNA (Teng, Usman,Frederick, & Wang, 1988). Molecular modeling studies havebeen carried out in order to completely understand theinteraction of various rotameric, tautomeric and ionizationstates of Hoechst 33258 (existing at physiological pH) withthe Dickerson-Drew dodecamer d(CGCGAATTCGCG)2sequence. The electrostatic and hydrogen bonded interac-tions (Figure 1) are found to be the major forces responsiblefor the Hoechst-DNA complexation. QM/MM studies revealimportant geometrical and energetic aspects of minorgroove binding. The high complexation energy (-549.67 kcalmol% 1) signifies that a stable complex is formed between

Hoechst and DNA. As a result of our study, the binding ofHoechst 33258 to the Dickerson–Drew sequence can be bet-ter understood and the results presented may be used indesigning of novel DNA binding drugs with useful biologicalapplications.

References

Teng, M.-K., Usman, N., Frederick, C. A., & Wang, A. H.-J. (1988). Themolecular structure of the complex of Hoechst 33258 and the DNAdodecamer d(CGCGAATTCGCG). Nucleic Acids Research, 16(6),2671–2690.

57. In-silico assessment of adenosinereceptor ligand binding domain: anew insight into adenosine receptorbased therapeutics

Navina Panneerselvana, Radha Madendranb andMalathi RangunathanaaDepartment of Genetics, Dr.ALM PG IBMS, University of Madras,Taramani 600113, Chennai; bDepartment of Bioinformatics, Vel’sUniversity, Velan Nagar, Pallavaram 600117, Chennai

[email protected]

Extracellular adenosine acts as a local modulator with a gen-erally cytoprotective function in the body. During ischemiaor inflammation conditions adenosine levels are elevated.Among the four adenosine receptors (ARs), A2b ARexpressed highly in angiogenesis during wound healing andtumor growth; its role in genetic and pharmacological stud-ies are known to an extend (Du et al., 2015), however thebinding ability of A2b with anti-angiogenic plant compoundsare not well known (Jacobson & Gao, 2006). In order to valid-ate the usefulness of this receptor model, docking analysis ofseveral selective and nonselective compounds were carriedout to find the binding affinities and selectivity of ligands toadenosine binding region of A2b AR. Plant compounds likeChrysin, Apigenin, Theobromine, Theophylline, Genistein,Resveratrol, Pentoxifylline and synthetic compounds DAPT,SU5416, SQ22536, ZM 241385 and MSR 1754 that are knownto be AR targets. Our results illustrated that A2b AR possessthe strongest interaction to Chrysin with higher binding freeenergy score of % 8.09 kcal/mol with 6-OH bond interactionsat ALA82, PHE84, VAL85, ALA60, ALA64 compared to positivecontrol of ZM241385 with % 8.70 kcal/mol binding energy.Other plant molecules like resveratrol make three electro-static interaction with GLN6, GL4, LEU2 with binding affinityof % 7.04 kcal/mol of OH bond; Theobromine and theophyl-line similarly showed lower binding affinity of % 6.448 kcal/mol and -6.41 kcal/mol. Apigenin shows % 6.76 kcal/mol with6-OH bond at THR5, LYS269, TRP270 electrostatically;Genistein shows % 6.33 kcal/mol binding affinity made of 5-OH bond with LEU2 (one bonding distance), GLN6 (fourbonding distance); and pentoxyflline with % 6.81 kcal/mol(OH bonding) distance of 2.3 at THR139. Among theFigure 1. Hydrogen bonding between Hoechst 33258 and DNA.


synthetic molecule DAPT with % 8.35 kcal/mol binding energymaking two strong OH at ASP7 two bond distance of 2.1and 2.0; next SU5416 with -7.218 kcal/mol with TRP270 andLYS269 OH bond. Furthermore, molecular stimulation ana-lysis might be carried out to find binding ability of the com-pounds tested. Based on the study result, the selected plantcompounds like chrysin might considered as novel and sig-nificant compound that can be modeled and characterizedfor auxiliary therapeutic implement (Figure 1).

Funding

This research has been supported by UGC-BSR award No: F-7-115/2007.

References

Du, X., Ou, X., Song, T., Zhang, W., Cong, F., Zhang, S., & Xiong, Y. (2015).Adenosine A2B receptor stimulates angiogenesis by inducing VEGFand eNOS in human microvascular endothelial cells. ExperimentalBiology and Medicine, 240(11), 1472–1479.

Jacobson, K. A., & Gao, Z. G. (2006). Adenosine receptors as therapeutictargets. Nature Reviews Drug Discovery, 5(3), 247.

58. Integration of binding potencyestimations and stability assessmentsfor therapeutic design against MurGof H. pylori

Pasala Chiranjeevi, Katari Sudheer Kumar,Nalamolu Ravina Madhulitha, Sharon Priya Alexanderand Amineni UmamaheswariBioinformatics Centre, Department of Bioinformatics, SVIMSUniversity, Tirupati 517507, Andhra Pradesh, India

[email protected]

The adverse effects and widespread emergence of resistantHelicobacter pylori strains to the existing drugs is becoming aserious public health concern. As a result there is a real andpressing demand to develop new therapeutics; however, itremains one of the major challenges to science and unmet

needs in the clinics. We have therefore selected UDP-N-acetylglucosamine–N-acetylmuramyl-(pentapeptide) pyro-phosphoryl-undecaprenol N-acetylglucosamine transferase(MurG), one of the promiscuous common targets for multipleH. pylori strains identified in our previous study (Pasala et al.,2018). Cell wall-related MurG is of special interest, since, it isinvolved in peptidoglycan biosynthesis with the potentialinteraction between other proteins, unique to the bacteria,has no mammalian counterpart, and agents inhibiting itssynthesis have the potential to become broad-spectrum ther-apeutics. In the study, the selected target was allowed toModeller9v20 to generate 100 models with incorporatingsubstrate Uridine-Diphosphate-N-Acetylglucosamine (UD1)using a template (PDB: 3S2U). After validations, the thirdmodel was deposited at Protein Model DataBase(PM0081627). The reviewed previous ligands of MurG wereallowed to hierarchical-clustering. Subsequently, the obtainedfive diverse ligands from five clusters with clustering strain1.261and DU1 were subjected to SwissSimilarity web-tool toperform ligand-based virtual screening of several libraries of3 billion small molecules. The retrieved 4177 similar contourswere further assigned to lead-optimization campaignincludes, rigid receptor docking (Glide parameters), quan-tum-polarized ligand docking (quantum mechanics) andinduced-fit docking (80 replications with charge calculations)coupled with Molecular Mechanics/Generalized Born SurfaceArea methods to estimate relative binding affinity (DGbind) ofthe previous, screened compounds and DU1. As a result,seven compounds were observed with better DGbind freeenergies than top diverse ligand BMS-190134 (% 63.12 kcal/mol) and DU1 (% 49.33 kcal/mol). Moreover, interaction stabil-ity of DU1 and the best lead1 (% 67.85 kcal/mol) with MurGwas dynamically assessed for 50 ns and 150 ns chemical timerespectively (Katari et al., 2016; Pasala et al., 2018b).Consequently, the consistency of lead1-MurG was found withlower deviations, fluctuations and fair compactness thanDU1-MurG. The stable interactions with lead1 have beenmaintained throughout the simulations period and correlatedwith docking analysis thereby that contacts might be vitalhotspots for catalytic activity of MurG. Therefore, as the pro-posed 7 leads fulfilling all the criteria from different aspectsof drug design study, these could be promiscuous thera-peutic agents against H. pylori (Figure 1).

Figure 1.


Acknowledgments

PC acknowledges to ICMR, New Delhi, for supporting with the SeniorResearch Fellowship (3/1/3/JRF-2014/HRD-8). Authors are highly thankfulto DBT, Ministry of Science and Technology, Govt. of India for providingBioinformatics infrastructure facility to SVIMS Bioinformatics Centre (BT/BI/25/037/2012 (BIF-SVIMST)).

References

Katari, S. K., Natarajan, P., Swargam, S., Kanipakam, H., Pasala, C., &Umamaheswari, A. (2016). Inhibitor design against JNK1 through e-pharmacophore modeling docking and molecular dynamics simula-tions. Journal of Receptors and Signal Transduction Research, 36(6),558–571.

Pasala, C., Chilamakuri, C. S. R., Katari, S. K., Nalamolu, R. M., Bitla, A. R., &Umamaheswari, A. (2018). An in silico study: Novel targets for poten-tial drug and vaccine design against drug resistant H. pylori. MicrobialPathogenesis, 122, 156–161.

Pasala, C., Chilamakuri, C. S. R., Katari, S. K., Nalamolu, R. M., Bitla, A. R., &Umamaheswari, A. (2018). Epitope-driven common subunit vaccine

design against H. pylori strains. Journal of Biomolecular Structure andDynamics. doi:10.1080/07391102.2018.1526714. (In press)

59. Interaction of noscapine withhuman serum albumin (HSA):a spectroscopic and molecularmodeling approach

Heerak Chugh and Ramesh ChandraDepartment of Chemistry, University of Delhi, New Delhi 110007,India [email protected]

Noscapine, an opium derived anticancer agent manifeststherapeutic applications and is compliant to beneficial modi-fications. In this paper, we study its interaction with humanserum albumin (HSA) by different spectroscopic methodsincluding UV–vis, fluorescence, circular dichroism and

Figure 1.


thermal denaturation and molecular docking. Binding andthermodynamics parameters support a feasible interaction ofNos-HCl with HSA. Further, type of interactions between Nos-HCl and HSA were also elucidated. The energy transferbetween HSA and Nos-HCl was analyzed by Forster mechan-ism and lifetime measurements. Additionally, it was shownthat Nos-HCl binds HSA in subdomain IIIA which constitutethe drug binding Sudlow’s Site II. Preliminary results for bind-ing site were deciphered via the site marker competitiveexperiments which were then confirmed using moleculardocking analysis.

References

Empey, D. W., Laitinen, L. A., Young, G. A., Bye, C. E., & Hughes, D. T. D.(1979). Comparison of the antitussive effects of codeine phosphate20mg, dextromethorphan 30mg and noscapine 30mg using citricacid-induced cough in normal subjects. European Journal of ClinicalPharmacology, 16(6), 393–397.

Mahmoudian, M., & Rahimi-Moghaddam, P. (2009). The anti-cancer activ-ity of noscapine: A review. Recent Patents on anti-Cancer DrugDiscovery, 4(1), 92–97.

Pasquier, E., & Kavallaris, M. (2008). Microtubules: A dynamic target incancer therapy. IUBMB Life, 60(3), 165–170.

60. Magnetic resonance spectroscopy-based metabolomics analysis ofposterior cingulate in patients withalzheimer’s disease: a systematicreview study

Kiarash Shirbandia,b, Mohammad Davoodic, ElmiraHosseinid, Fakher Rahime,f and Babak ArjmandfaNeuroimaging Network (NIN), Universal Scientific Education andResearch Network (USERN), Ahvaz, Iran; bDepartment of Radiologic

Technology, Ahvaz Jundishapur University of Medical Sciences,Ahvaz, Iran; cDepartment of Radiology, Shariati Hospital, TehranUniversity of Medical Sciences, Tehran, Iran; dNeuroimagingNetwork (NIN), Universal Scientific Education and ResearchNetwork (USERN), Tehran, Iran; eHealth Research Institute,Thalassemia and Hemoglobinopathies Research Centre, AhvazJundishapur University of Medical Sciences, Ahvaz, Iran;fMetabolomics and Genomics Research Center, Endocrinology andMetabolism Molecular-Cellular Sciences Institute, Tehran Universityof Medical Sciences, Tehran, Iran

Posterior cingulate cortex (PCC) is a paralympic cortical struc-ture that is located in the middle of default mode network(DMN) that has a fundamental role in connecting differentregions of the DMN (Hampson, Driesen, Skudlarski, Gore, &Constable, 2006). The aim of this meta-analysis was to assessthe metabolomics content changes in PCC of the patientswith Alzheimer disease (AD) comparing to healthy controls(HC). We performed a comprehensive search through eightinternational indexing databases, including PubMed, Scopus,Cochrane library, CINAHL, ISI Web of Science, Science Directfrom inception, Embase and PROSPERO from 1980 to 2018.Two authors independently extracted data and classifiedmethods for analysis. Overall, a total of 3,067 relevant studieswere initially found of which 18 studies comprising 1,647cases, (658 (40%) males and 941 (60%) females, 921 (55.9%)HC and 678 (44.1%) AD cases). The analyses showed signifi-cant differences between AD patients and HC in term of

Figure 1. Overview of the role of various metabolites engaged in the metabolicpathway of Alzheimer disease (AD).


NAA (NAA: N-Acetyl Aspartate), mI (myo-Inositol), Glu(Glutamine) and Glx (Glutamate & Glutamine) concentrations.Also, metabolite ratios including NAA/Cr, Cho/Cr and mI/Crwere significantly different. Hence, there were no significantdifferences in term of Cr and Cho levels and mI/NAA ratiobetween AD and HC groups. Our meta-analysis showedmetabolite changes in the PCC can be used as a marker forearly diagnosis of the AD. Although NAA biomarker is con-sidered to be the most important metabolite and mI is mostsensitive for early diagnosis of the AD, Cho/Cr ratio as a crit-ical biomarker can be of interest to researchers (Figure 1).

References

Hampson, M., Driesen, N. R., Skudlarski, P., Gore, J. C., & Constable, R. T.(2006). Brain connectivity related to working memory performance.Journal of Neuroscience, 26(51), 13338–13343.

61. Minimizing toxicity andmaximizing the potency of bioderivedcompounds by syntheticfragmentation—a computational‘proof-of-concept’

Fisayo A. Olotu, Geraldene Munsamy andMahmoud E. SolimanMolecular Bio-computation and Drug Design Laboratory, School ofHealth Sciences, University of KwaZulu Natal, Westville Campus,Durban 4001, South Africa [email protected]

Despite the potency embedded in natural products withregards to multiple disease treatment, the numerous chal-lenges of toxicities and undesirable biological ‘off-targeting’have limited its clinical transition. More recently, the syn-thetic reduction of complex natural products into simplerfragments has been identified as a viable strategy to developnext generation leads with improved potencies and minimaltoxic effects. Therefore, to validate the efficacy of thismethod, we employed combinatorial molecular modeling

and cheminformatics techniques to describe the differentialpharmacological and antagonistic activities of a selected frag-ment, SB640 and its parent compound, Anguinomycin Dtowards their target protein, Exportin Chromosome RegionMaintenance 1 (CRM1), involved in pro-carcinogenic chemo-therapeutic resistance. Our findings revealed that the fragmentexhibited improved pharmacokinetics with minimal toxicitiesand off-target activities compared to the parent compound.Furthermore, reduction into a smaller fragment enabled opti-mal positioning and binding with crucial residues at the pro-tein target site which in turn accounted for a more prominentCRM1 inactivation as compared to the parent compound thathad minimal structural effects due to motion and dynamicalconstraints caused by its long polyketide tail. Our findingstherefore indicate that the ‘size does not matter’ and thatreduction of complex bioderived compounds to fragmentscould be an essential strategy for improving potency and mini-mizing associable adverse drug reactions (Figure 1).

References

Crane, E. A., & Gademann, K. (2016). Capturing biological activity in nat-ural product fragments by chemical synthesis. Angewandte ChemieInternational Edition, 55, 2–23.

Olotu, F. A., Munsamy, G., & Soliman, M. E. (2018). Does size really mat-ter? Probing the efficacy of structural reduction in the optimization ofbioderived compounds—a computational “proof-of-concept”.Computational and Structural Biotechnology Journal, 16, 573–586.

62. Mutant p53 REs and themechanism of gain of functionin cancer

Jessy Safieh, Demitrij Golovenko and Tali E. HaranFaculty of Biology, Technion, Haifa, Israel [email protected]

p53 functions as a sensor of cellular stress signals. A varietyof stress signals stabilize its structure and bring about anincrease in its concentration, with the end result of a varietyof functional outcome preventing cancer. However, p53 itself

Figure 1.


is mutated in 50% of all cancer cases. p53 core domain isthe main target for mutations in human tumors. 95% of themutations identified in human tumors are found in this region.This underscores the importance of sequence-specific DNAbinding by the core for p53 functions. Gain-of-function muta-tions are mutations that switch p53 into a tumor-promotingoncogene, by endowing the mutant protein with new abilities,activating new target genes, that can contribute to variousstages of tumor progression. Multiple mechanisms were pro-posed to account for different mutant p53 gain-of-functionactivities. A major transcriptional mechanism for gain-of-func-tion of mutant p53 is binding of mutant p53 through cooper-ation with other transcription factors. We will present a novelmodel for mutant p53 gain-of-function in cancer that recon-ciles many observations on the gain-of-function phenomenon.We will also present binding data demonstrating that the roleplayed by DNA structure in mutant p53/DNA interaction fol-lows the rules established for wild-type p53/DNA interactions.

63. How the tumor suppressor p53recognizes its DNA response elements

Dmitrij Golovenkoa,b, Oksana Degtjarika, Tali E.Haranb, Haim Rozenberga and Zippora ShakkedaaDepartment of Structural Biology, Weizmann Institute of Science,Rehovot, 76100, Israel; bDepartment of Biology, Technion–IsraelInstitute of Technology, Haifa, 32000, Israel

[email protected]

In response to cellular stress signals, the tumor suppressorp53 acts as transcription factor by binding as a tetramer to awide range of DNA sites, thereby activating numerous genesthat are critical for cancer prevention (Vogelstein, Lane, &Levine, 2000). Its main functional domain, the DNA bindingcore domain (p53DBD), is accompanied by structured tetra-merisation domain and intrinsically disordered N- and C-ter-mini. p53 binds specifically to double-stranded DNAresponse elements (REs) which are composed of two deca-meric half-sites of the general form RRRCWWGYYY (R ¼ A, G;W ¼ A, T; Y ¼ C, T) (el-Deiry, Kern, Pietenpol, Kinzler, &Vogelstein, 1992). Our previous studies on crystal structuresof p53DBD in complexes with consensus DNA sites revealednon-canonical Hoogsteen (HG) base pairs at the A/T doubletswithin conserved CATG motifs at the half-site centers,flanked by GGG/CCC (Kitayner et al., 2010). In the HG geom-etry, the A bases are rotated around their glycosidic bondsby nearly 180# relative to that of the common Watson-Crick(WC) geometry, to form alternative hydrogen bonds (Figure1(a)). As a result of the HG geometry, the backbone atomson opposite DNA strands are closer by " 2 Å in the region ofHG base pairs compared to that of WC base pairs, affectingthe shape of the DNA helix. This finding led to the proposalthat the unique DNA shape enhances the stabilization of thep53DBD-DNA complex (Kitayner et al., 2010).

To characterize the effect of DNA shape on p53-DNAinteractions in terms of structural and biochemical aspects,we used a novel approach to ‘lock’ base-pairing into WC orHG geometry. The method relies on designing REs where thecentral A/T doublets are replaced by modified nucleotides in

Figure 1. Schematic representation of Watson-Crick and Hoogsteen base pairs for natural and modified variants. Hydrogen bonds between donor and acceptorgroups indicated by blue bars, and repulsive interactions by red bars.


a way that avoids introduction of bulky groups and preservesthe A/T patterns at the minor-groove edges of the modifiedbase-pairs in both geometries. Thus, to shift the equilibriumtoward the HG geometry, A bases were replaced by 2-oxo-A(2OA), and to enforce the WC geometry, A and T bases werereplaced by Inosine (I) and 5-methyl-C (5mC), respectively(Figure 1(b,c)). In this manner, the selection between the twoforms, HG or WC, is determined by the balance betweenattractive and repulsive interactions, as manifested by thehigh-resolution crystal structures of the two types of p53-DNA complexes, displaying the expected conformational dif-ferences between the two helices (Figure 2(a,b)). A detailedanalysis of the various structures combined with DNA bind-ing and kinetic studies demonstrated that complexes withthe unusual HG geometry are stabilized relative to thosewith all-WC base pairs. We also showed that two naturalhigh-affinity REs, related to DNA-damage response andincorporate CATG motifs, are also predisposed to adopt theunique DNA shape induced by HG base pairs (Golovenkoet al., 2018).

To conclude, the combined structural and biochemicaldata demonstrate that in addition to direct readout made bydirect interactions between the protein and DNA, indirectreadout rendered by shape readout plays a major role inDNA recognition by proteins.

References

el-Deiry, W. S., Kern, S. E., Pietenpol, J. A., Kinzler, K. W., & Vogelstein, B. (1992).Definition of a consensus binding site for p53. Nature Genetics, 1(1), 45–49.

Golovenko, D., Brauning, B., Vyas, P., Haran, T. E., Rozenberg, H., & Shakked,Z. (2018). New Insights into the role of DNA shape on its recognition byp53 Proteins. Structure (London, England: 1993), 26(9), 1237–1250.

Kitayner, M., Rozenberg, H., Rohs, R., Suad, O., Rabinovich, D., Honig, B.,& Shakked, Z. (2010). Diversity in DNA recognition by p53 revealed bycrystal structures with Hoogsteen base pairs. Nature Structural &Molecular Biology, 17, 423–429.

Vogelstein, B., Lane, D., & Levine, A. J. (2000). Surfing the p53 network.Nature, 408(6810), 307–310.

64. Structural studies on reactivationof tumor-related p53 mutants bymethylene quinuclidinone (MQ), theactive drug spontaneously formedfrom APR-246

Oksana Degtjarika, Dmitrij Golovenkoa, Yael Diskin-Posnerb, Lars Abrahms"enc, Haim Rozenberga andZippora ShakkedaaDepartment of Structural Biology, Weizmann Institute of Science,Rehovot 76100, Israel; bDepartment of Chemical Research Support,Weizmann Institute of Science, Rehovot 76100, Israel; cApreaTherapeutics AB, Solna, 17165, Sweden

[email protected]

The tumor suppressor p53 is also known as the ‘guardian ofthe genome’ due to its ability to act as a transcription factorthat regulates the expression of a range of target genes inresponse to genotoxic stress, leading to DNA repair, cell cyclearrest or apoptosis, all essential to prevent cancer (Vogelstein,Lane, & Levine, 2000). The function of p53 can be compro-mised by mutations that lead to p53 inactivation and thus tocancer development. The majority of these mutations arelocated at the DNA binding core domain of p53 (p53DBD).Among them, six mutational ‘hotspots’ at residues: R175, G245,R248, R249, R273 and R282 (Figure 1), were shown to occur athigh frequency in human cancer (Olivier et al., 2002). The cor-responding amino-acid replacements (shown in red) can eitheraffect p53 binding to DNA and referred as DNA-contactmutants: R273H/C, R248Q/W; or cause conformational changesin p53DBD and referred as structural mutants: R175H, G245S,R249S and R282W. Several crystal structures of the p53DBD ofsuch mutants have been reported (Eldar, Rozenberg, Diskin-Posner, Rohs, & Shakked, 2013; Suad et al., 2009).

PRIMA-1 and APR-246/PRIMA-1MET are small moleculesthat are converted into the biologically active compound,methylene quinuclidinone (MQ) which reactivates mutantp53 by binding covalently to cysteines in the p53DBD(Lambert et al., 2009). APR-246 is presently undergoing clin-ical trials (https://www.clinicaltrials.gov). In our study, weinvestigate the reactivation mechanisms of mutant p53 by MQfrom a series of high-resolution crystal structures of wild-typep53 and several hotspot mutants bound to MQ in the absenceand/or the presence of DNA. Our data show that MQ can bind

Figure 2. Structures of p53DBD tetramers bound to DNA REs (20 bp long) with4 base pairs ‘locked’ into Hoogsteen geometry (A) or Watson-Crick geometry(B) (highlighted by thick lines).


to several cysteine residues located at the surface of the coredomain at positions: 124, 182, 229, 275 and 277. A detailedcomparison between the structures of specific p53 mutantsbefore and after binding to MQ, reveals the role played by MQin stabilizing p53 and its interaction with DNA, thus providinga structural framework for the design of new molecules forspecific targeting of cysteines in p53 mutants.

References

Eldar, A., Rozenberg, H., Diskin-Posner, Y., Rohs, R., & Shakked, Z. (2013).Structural studies of p53 inactivation by DNA-contact mutations andits rescue by suppressor mutations via alternative protein-DNA inter-actions. Nucleic Acids Research, 41(18), 8748–8759.

Kitayner, M., Rozenberg, H., Kessler, N., Rabinovich, D., Shaulov, L., Haran,T. E., & Shakked, Z. (2006). Structural basis of DNA recognition by p53tetramers. Molecular Cell, 22(6), 741–753.

Lambert, J. M., Gorzov, P., Veprintsev, D. B., Soderqvist, M., Segerback, D.,Bergman, J., … Bykov, V. J. (2009). PRIMA-1 reactivates mutant p53by covalent binding to the core domain. Cancer Cell, 15(5), 376–388.

Olivier, M., Eeles, R., Hollstein, M., Khan, M. A., Harris, C. C., & Hainaut, P.(2002). The IARC TP53 database: New online mutation analysis andrecommendations to users. Human Mutation, 19(6), 607–614.

Suad, O., Rozenberg, H., Brosh, R., Diskin-Posner, Y., Kessler, N., Shimon,L. J., … Shakked, Z. (2009). Structural basis of restoring sequence-specific DNA binding and transactivation to mutant p53 by suppres-sor mutations. Journal of Molecular Biology, 385(1), 249–265.

Vogelstein, B., Lane, D., & Levine, A. J. (2000). Surfing the p53 network.Nature, 408(6810), 307–310.

65. Potent MMP-14 antagonist designthrough screening, docking anddynamics studies

Katari Sudheer Kumar!, Pasala Chiranjeevi,Nalamolu Ravina Madhulitha, Vankadoth UmakanthNaik and Amineni UmamaheswariBioinformatics Centre, Department of Bioinformatics, SVIMSUniversity, Tirupati 517507, Andhra Pradesh, India

[email protected]!Presenting author: Email: [email protected]

Matrix metalloproteinase-14 (MMP-14) or membrane typeMMP-1 is an extra cellular matrix (ECM) protease activatesprogelatinase A (MMP-2) involved in proteolysis of collagen,gelatin, elastin, laminin, fibronectin and integrins for cancer-ous cell to invade and metastasise. About 2,756 inhibitorsfrom CLRI-MMpi database were retreived, optimized andminimized through LigPrep using OPLS3 forcefield. Theligands were passed through virtual screening workflow(VSW) protocol, where the ligands were refined and dockedby high throughput virtual screening [HTVS-10%], standardprecision [SP-10%] and extra precision [XP-100%] over theGrid of MMP-14 (5H0U) using grid-based ligand docking withenergetics (GLIDE) and binding free energy (DG) was esti-mated by Prime-MM/GBSA for 27 dock complexes(Chiranjeevi, Swargam, Pradeep, Hema, & Kumar, 2016). Thebest bound MMpi database inhibitors (MMpI1999248,MMpI2009210 and MMpI200383) were screened againstSwissSimilarity databases comprising " 0.3 Billion compoundsresulted 2756 structural analogs. 28 compounds wereobtained from VSW, fourteen compounds were better scored(XP G and DG) than the best three MMpi. These wereredocked by quantum polarized ligand docking (QPLD) andresulted 5 leads; the best lead (lead 1) possesses QPLD XPGscore of % 8.711 kcal/mol and DG score of -% 67.035 kcal/mol, but the best MMpi (MMpI1999248) possesses QPLD XPGscore of -6.092 kcal/mol and DG score of -66.538 kcal/mol(Madhulitha, Pradeep, Sandeep, Hema, & Chiranjeevi, 2017).The best lead and the best MMpi (MMpI1999248) MMP-14complexes were simulated for 100 ns and the dynamics wereassessed for stability by analyzing root-mean-square-devi-ation (RMSD), root-mean-square-fluctuation (RMSF), totalenergy (T.E.), potential energy (P.E.), protein-ligand contacts(hydrogen bond, pi-cation, hydrophobic, metal co-ordinationand water bridges), radius of gyration and torsions(Katari et al., 2016). Lead 1-MMP-14 complex showed T.E. and

Figure 1. Six hotspot mutation sites in p53DBD bound to DNA. p53 binds to its DNA target as a tetramer (dimer of dimers). Here is a view of a p53DBD dimer (grey)bound to DNA (blue), showing the six hotspot sites (red). The view is along the DNA helix. The figure is based on coordinates from PDB code 2ac0 (Kitayner et al., 2006).


P.E. of % 64,432.622 kcal/mol and % 78,558.567 kcal/mol lesserthan MMpI1999248-MMP-14 complex with % 64,405.771 kcal/mol and % 78,552.540 kcal/mol. Lead 1 with respective to pro-tein and lead 1 with respective to lead 1: average RMSD was2.16 Å and 0.68 Å, average RMSF was 0.99 Å and 0.38 Å lesserthan the MMpI1999248 with respective to protein and withrespective to itself: average RMSD was 5.50 Å and 2.75 Å andaverage RMSF of 2.26 Å and 1.85 Å were observed during100 ns molecular dynamics (MD) simulations. The overallmolecular contacts formed by MMP-14-lead 1 (9,745) arecomparatively higher than MMP-14-MMpI1999248 (9,482) in

100 ns MD simulations suggested that lead 1 could act as apotent antagonist by blocking the functional activity ofMMP-14, MMp-2 activation, ECM degradation, cancer cellinvasion as well as metastasis.

Acknowledgments

KSK is highly acknowledged to DBT for sanctioning JRF and SRF (BT/BI/25/037/2012). Authors are thankful to DBT, Ministry of Science andTechnology, Government of India, New Delhi for supporting the workthrough BTISnet BIF program (No. BT/BI/25/037/2012).


References

Chiranjeevi, P., Swargam, S., Pradeep, N., Hema, K., & Kumar, K. S.(2016). Inhibitor design for VacA toxin of Helicobacter pylori. Journalof Proteomics and Bioinformatics, 9, 220–225. doi: 10.4172/jpb.1000409.

Katari, S. K., Natarajan, P., Swargam, S., Kanipakam, H., Pasala, C., &Umamaheswari, A. (2016). Inhibitor design against JNK1 through e-pharmacophore modeling docking and molecular dynamics simula-tions. Journal of Receptor and Signal Transduction Research, 36(6),558–571.

Madhulitha, N. R., Pradeep, N., Sandeep, S., Hema, K., & Chiranjeevi, P.(2017). E-Pharmacophore model assisted discovery of novel antago-nists of nNOS. Biochemistry & Analytical Biochemistry, 6, 307.doi:10.4172/2161-1009.1000307

66. Scaffold hopping strategy on theroute discerning novel glutathioneperoxidase agonists

Natarajan Pradeepa, Manne Munikumarc, SandeepSwargamd, Kanipakam Hemae, Katari Sudheerkumarb, Amineni Umamaheswarib, Praveen KumarGuttulaa and Mukesh Kumar Guptaa

aBioinformatics Centre, Department of Biotechnology and MedicalEngineering, National Institute of Technology, Rourkela, Odisha769008, India; bBioinformatics Centre, Department ofBioinformatics, Sri Venkateswara Institute of Medical ScienceUniversity, Tirupati 517507, Andhra Pradesh, India; cNIN-TATACentre for Excellence in Public Health Nutrition, ICMR-NationalInstitute of Nutrition, Jamai-Osmania (Post), Hyderabad 500007,Telangana, India; dJamia Hamdard Institute of Molecular Medicine,Jamia Hamdard University, Hamdard Nagar, New Delhi 110062,India; eFunctional genomics unit, CSIR-IGIB, New Delhi 110007,India [email protected]

Alzheimer’s disease (AD), the utmost common and progres-sive neurodegenerative disease characterized by aggregationof b-amyloid plaques triggered by mitochondrial oxidativestress also. Raising substantial evidences evokes that dilutedantioxidant activity correlates with overproduction of freeradicals and peroxides. Which extends the alterations inmembrane permeability damaging the mitochondrial respira-tory chain, subsequently amplifying the neuronal dysfunctionthere by triggering neurodegeneration. Glutathione peroxi-dases (GPx1-2) is abundant antioxidant enzyme that catalyzereduction of hydrogen peroxide to water. Increased free radi-cals and absurdity in potentiality to detoxify the reactive

Figure 1.

Figure 2.


intermediates associated with diluted activity of GPx has madeit as a possible attractive therapeutic target for AD interven-tion. Five existing agonists were subjected to geometry basedvirtual screening against in-house library having more than 21million small molecules. The resulted 1273 hits were subjectedto rigid receptor docking (RRD) and quantum polarized liganddocking (QPLD) succeeded with binding free-energy (DG) cal-culations, resulted best ligands (2 for GPx1 and 1 for GPx2).The best ligands and existing agonists were subjected to scaf-fold hopping against fragment libraries resulted 644 com-pounds, which are subjected to multi-level multiple dockingstrategies to derive the best leads (12 for GPx1; 14 for GPx2)with favorable pharmacokinetic properties (Figure 1). Further,GPx and the best lead complexes were subjected to 50 ns MDsimulations to analyze the stability (Figure 2) as well as theintactness (Figure 3) of the best leads towards the receptor.The proposed leads, obtained from scaffold hopping of exist-ing agonists showed favorable binding orientation withincreased binding affinity towards GPx, by enhancing the sulf-hydryl groups availability to ROS and peroxide intermediatesensures the anti-oxidant activity. The intensifying GPx levelswith the proposed leads enriches the anti-oxidant effect. Thus,the results emphasis that the proposed novel leads would beeffective in treating oxidative stress mediated AD therapeutics.

References

Natarajan, P., Priyadarshini, V., Pradhan, D., Manne, M., Swargam, S.,Kanipakam, H., … Amineni, U. (2016). E-pharmacophore-based virtualscreening to identify GSK-3b inhibitors. Journal of Receptor and SignalTransduction Research, 36(5), 445–458.

Pradeep, N., Munikumar, M., Swargam, S., Hema, K., Sudheer Kumar, K., &Umamaheswari, A. (2015). 197 Combination of e-pharmacophore

modeling, multiple docking strategies and molecular dynamic simula-tions to discover of novel antagonists of BACE1. Journal ofBiomolecular Structure and Dynamics, 33(supp1), 129–130.

Wang, L., Deng, Y., Wu, Y., Kim, B., LeBard, D. N., Wandschneider, D., …Abel, R. (2017). Accurate modeling of scaffold hopping transforma-tions in drug discovery. Journal of Chemical Theory and Computation,13(1), 42–54.

67. The distribution of 3,5,30-triiodothyronine between thetransport systems of blood and nucleiof the tissues

Margarita I. Garipova, Alfira I. Shigapova, Rashit G.Farkhutdinov, Vadim V. Fedyaev, Julia M. Sotnikovaand Alfira B. YakupovaDepartment of Biology, Bashkir State University, Z. Validy, 32, Ufa450076, Russian Federation [email protected]

It is known that hydrophobic hormones can be transportingin blood by two mechanisms: in serum proteins complexand in internal volume of erythrocytes (Dolomatov et al.,1999; Eliseeva et al., 2009; Garipova & Usmanova, 2013;Garipova & Dazko, 2015; Garipova, Morugova, Kireeva,Ibragimov, & Baranova, 2010; Gimatdinova et al., 2011). It isinteresting to answer the question of how the intracellularhormone concentration is related to it is concentration ineach of hormone transporting systems. There distribution of3,5,30-triiodothyronine (T3) between the transport systems ofblood and nuclei of leucocytes, lung and liver tissues of rat

Figure 3.


were examined. The 3,5,3’-triiodothyronine concentrations inblood plasma, hemolysate and nucleoplasm prepared from leu-cocytes, lung and liver of 10 laboratory non-linear male ratswere determined by enzyme immunoassay. The 3,5,30-triiodo-thyronine concentration in blood plasma was averaged6.33±0.34pmol/L. The T3 concentration in erythrocyte internalvolume (hemolysate) was reliably less% 2.94pmol/L (! ¼ 8, 34,h ¼ 0.001).This indicates relative depletion of erythrocyte tri-iodothyronine depot. T3 concentrations in tissue nucleus weredramatically more, than in blood hormone transporting systems.The triiodothyronine concentration in leukocytenucleiwas 2.57times more than in blood plasma (16.24±0.98pmol/L). Inlungnuclei T3 concentration was 12.23±0.87pmol/L and in livernucleoplasm 16.68±1.03pmol/L. Perhaps, triiodothyronine con-centration is associated with biosynthetic activity of the tissue:correlation coefficient between T3 and RNA concentrations innucleoplasm was 0.57 (h ¼ 0.003).

The distribution of triiodothyronine between bloodplasma, internal volume of erythrocytes and blood leukocytenuclei of healthy donors were studied. It’s shown, that inblood plasma T3 concentration is 4.14 ± 0.6 pmol/L, that is17.6% of total hormone content in blood sample. The tri-iodothyronine concentration in erythrocytes was6.05 ± 0.4 pmol/L, that is, 25.8% of total hormone content. Itis noteworthy, that the T3 concentration in leucocyte nucleo-plasm averaged 13.3 ± 0.6pmol/L, that is more than threetimes higher than the hormone concentration in bloodplasma. Thus, both in the nuclei of laboratory rats tissuesand healthy donors T3 concentration in nuclei significantlyexceeds the T3 concentration in both blood hormone transport-ing systems, which indicates the mechanisms of active transportand accumulation of 3,5,30-triiodothyronine in the tissue nuclei.

References

Dolomatov, S. I., Pishak, V. P., Slipenuk, T. S., Meshishen, I. F., &Okopnaya, T. V. (1999). Erythrocyte capacity to depose thyroid hor-mones: Regulatory function of physicochemical factors in vitro.Voprosy Medicinskoy Chimii, (6), 572–577.

Eliseeva, O. S., Kireeva, N. A., Pershina, A. S., & Garipova, M. I. (2009).Investigation of the insulin interaction with the erythrocytes surface.Vestnik OGU, N (6), 476–478.

Garipova, M. I., & Usmanova, R. R. (2013). Isolation and partial characteriza-tion of a general hormone transporting blood protein complex. Journalof Biomolecular Structure and Dynamics, 31(supp 1), 118. Vol. No.

Garipova, M. I., & Dazko, O. I. (2015). Two different hormone transporting sys-tems inhuman blood: Features of peptide hormone transport in humanblood. Journal of Biomolecular Structure and Dynamics, 33(S1), 162. Vol. No.

Garipova, M. I., Morugova, T. V., Kireeva, N. A., Ibragimov, R. I., & Baranova,M. V. (2010). Affinity preparation and insulin-binding human blood serumprotein diversity investigation. Voprosy Medicinskoy Chimii, (8), 40–44.

Gimatdinova, E. V., Hayrullina, R. M., Garipova, M. I., Sotnikova, U. M., &Veselov, S. U. (2011). Diagnostic and prognostic opportunities of pro-calcitonin and c-reactive protein in different infectious and inflamma-tory process with children. Fundamental Research 10(2), 280–282.

68. Using structure to identifyprotein–protein and drug proteininteraction networks

Barry Honig

Departments of Systems Biology and Biochemistry and MolecularBiophysics, Columbia University, NY 10032, USA

[email protected]

The presentation will describe the application of proteinstructure to predict protein–protein and protein-ligand inter-actions on a genome-wide scale. Protein networks and sig-naling pathways are constructed and described in structuralterms yielding multiple potential targets for drug interven-tion. Drug leads are generated via structure alignment of tar-get proteins with proteins that form a complex with smallmolecules where the structure is known. An integrated soft-ware package allows for the multi-directional navigationbetween small molecules, proteins and networks/pathwaysusing large genomic, structural and chemical databases.

Funding

This research has been supported by NIH (5-R01-GM030518-38).

References

Garzon, J. I., Deng, L., Murray, D., Shapira, S., Petrey, D., & Honig, B.(2016). A computational interactome and functional annotation forthe human proteome. eLife, 5, e18715.

Hwang, H., Dey, F., Petrey, D., & Honig, B. (2017). Structure-based predic-tion of ligand–protein interactions on a Genome-wide Scale.Proceedings of the National Academy of Sciences of the United States ofAmerica, 114(52), 13685–13690.

Hwang, H., Petrey, D., & Honig, B. (2016). A hybrid method for protein–-protein interface prediction. Protein Science, 25(1), 159–165.

Zhang, Q. C., Petrey, D., Deng, L., Qiang, L., Shi, Y., Thu, C. A., … Honig,B. (2012). Structure-based prediction of protein–protein interactionson a Genome-wide Scale. Nature, 490(7421), 556– 560.

Zhang, Q. C., Petrey, D., Garzon, J. I., Deng, L., & Honig, B. (2012). PrePPI:A structure-informed database of protein–protein interactions. NucleicAcids Research, 41(D1), D828–D833.

69. Applying quantitative singlemolecule localization microscopyto probe the mechanism ofnucleocytoplasmic transport

Kathleen M. Lennon, Devin L. Wakefield, Matthew S.Brehove, Ottavia Golfetto, Sunetra Biswas and TijanaJovanovic-TalismanDepartment of Molecular Medicine, Beckman Research Institute,City of Hope, CA 91010, USA [email protected]

Transport between the nucleus and cytoplasm is tightly regu-lated by nuclear pore complexes (NPCs). While these intricatemolecular machines obstruct passage of nonspecific biomole-cules, cargo-laden transport proteins are efficiently transportedacross the nuclear membrane. NPCs are essential for transcrip-tion, signaling and other fundamental cellular processes. Assuch, errors in the nucleocytoplasmic transport can cause cellu-lar dysregulation. Dysregulated transport has been observed inboth neurodegenerative disorders and cancer. Despite its criticalrole in health and disease, specific mechanism(s) of nucleocyto-plasmic transport currently remain unresolved. Understanding


the NPC transport mechanism(s) has been hindered by the (1)complexity of NPC geometry, (2) disordered nature of barrierelements called FG-Nups and (3) challenge of measuring milli-second transport events at nanometer scales in the intricate cel-lular environment. To probe potential transport mechanisms,we combined biochemical approaches with advanced quantita-tive single molecule localization microcopy (Golfetto et al.,2018). We measured both binding and diffusion of transportproteins on oriented FG-Nup monolayers. Our ultimate goal isto obtain critical information about both physiological andpathological NPC transport at the single molecule level.

Funding

This research has been supported by STOP CancerFoundation and Beckman Research Institute.

References

Golfetto, O., Wakefield, D. L., Cacao, E. E., Avery, K., Kenyon, V., Jorand,R., … Jovanovic-Talisman, T. (2018). A platform to enhance quantita-tive single molecule localization microscopy. Journal of the AmericanChemical Society, 140(40), 12785.

70. Self-regulating mechanisms of bi-directional transport through thenuclear pore complex

T. Zheng, C. Gu and A. ZilmanDepartment of Physics, Institute for Biomaterials and BiomedicalEngineering, University of Toronto, 60 St George st, Toronto, ON,Canada M1M 2P7 [email protected]

Nuclear pore complex (NPC) is a biomolecular ‘nanomachine’that controls nucleocytoplasmic transport in eukaryotic cells.The key component of the functional architecture of the NPC isthe assembly of the polymer-like intrinsically disordered pro-teins that line its passageway and play a central role in theNPC transport mechanism. Due to paucity of experimentalmethods capable to directly probe the morphology and thedynamics of this assembly in intact NPCs, much of our know-ledge about its properties derives from in vitro experimentsinterpreted through theoretical and computational modeling.Remarkably, despite their molecular complexity, much of the

behavior of these assemblies and their selective permeabilitywith respect to cargo-carrying transport proteins can be under-stood based on minimal complexity models relying on thestatistical physics of molecular assemblies on the nanoscale.

Due to the unstructured nature of the proteins in the NPCpassageway, it does not possess a molecular ‘gate’ that transi-tions from an open to a closed state during translocation ofindividual cargoes. Rather, its passageway simultaneously con-tains multiple transport proteins carrying different cargoes inboth directions. Although this feature increases NPC through-put, it remains unclear how the NPC maintains selective andefficient bi-directional transport under such crowded condi-tions. I will present of a coarse-grained computational modelof the NPC transport and will discuss various proposed solu-tions to the crowding problem in light of the model resultsand the available experimental data (Figure 1).

Funding

This research has been supported by National Science andEngineering Research Council of Canada through the NSERCDiscovery Grant Program.

References

Jovanovic-Talisman, T., & Zilman, A. (2017). Protein transport by thenuclear pore complex: Simple biophysics of a complex biomachine.Biophysical Journal, 113 (1), 6–14.

Vovk, A. (2016). Simple biophysics underpins collective conformations ofthe intrinsically disordered proteins of the nuclear pore complex.eLife, e10785

71. Statistical mechanical modelof transport receptor binding in thenuclear pore complex

Rob D. CoalsonDepartment of Chemistry, University of Pittsburgh, Pittsburgh, PA, USA

The nuclear pore complex (NPC) is a large protein complexthat controls the flow of proteins and mRNA into and out ofthe nuclei of eukaryotic cells. A major component of the NPCconsists of natively unfolded nucleoporin (nup) proteins,which are anchored on the inside of a cylindrical pore scaf-fold. They form a polymer brush that assists in the regulation

Figure 1.


of cargo flow from nucleus to cytoplasm. Large moleculesthat enter/leave the nucleus must attach themselves to spe-cial receptor proteins that ferry the cargo molecules in/outof nucleus. Via hydrophobic contacts between the receptorprotein and the nup filaments, the receptor proteins bindweakly and reversibly to the nup filaments and make theirway through the pore, via a mechanism that is at presentunknown. The complexity of the in vivo NPC motivates con-struction of coarse-grained models that can capture some ofits essential features while retaining computational tractabil-ity. We have developed such a model, focusing particularly onthe interactions of this polymer brush with solution phasenanoparticles that are attractive to brush monomers (mimick-ing the attraction of receptor proteins to hydrophobic seg-ments of nup filaments in the NPC). These attractions causethe nanoparticles to infiltrate into the polymer brush and alterits brush morphology. We have developed a Self-ConsistentField Theory model to analyze the equilibrium properties ofthe brush-nanoparticle system in an approximate but compu-tationally efficient manner (Opferman, Coalson, Jasnow, &Zilman, 2013). In addition, we have performed large scalecoarse-grained Molecular/Langevin Dynamics simulations toexplore the same properties (Nasrabad, Jasnow, Zilman, &Coalson, 2016; Ozmaian, Jasnow, Nasrabad, Zilman, & Coalson,2018). A variety of results pertaining to collapse/expansion ofthe brush upon nanoparticle infiltration will be presented.Their relevance to the in vivo mechanism of NPC operation willbe stressed (Vovk et al., 2016).

References

Nasrabad, A. E., Jasnow, D., Zilman, A., & Coalson, R. D. (2016). Precisecontrol of polymer coated nanopores by nanoparticle additives:Insights from computational modeling. Journal of Chemical Physics,145, 064901. 1-5.

Opferman, M. G., Coalson, R. D., Jasnow, D., & Zilman, A. (2013). Themorphology of polymer brushes infiltrated by attractive nanoinclu-sions of various sizes. Langmuir, 9, 8584–8591.

Ozmaian, M., Jasnow, D., Nasrabad, A. F., Zilman, A., & Coalson, R. D.(2018). Effects of cross-linking on partitioning of nanoparticles into apolymer brush: Coarse-grained simulations test simple approximatetheories. Journal of Chemical Physics, 148(2), 024902. 1-12.

Vovk, A., Gu, C., Opferman, M. G., Kapinos, L. E., Lim, R. Y. H., Coalson,R. D., Zilman, D. … (2016). Simple biophysics underpins collectiveconformations of the intrinsically disordered proteins of the nuclearpore complex. eLife, 5, 10785. 1–29.

72. The nuclear pore complex:paradoxes and possibilities

Roderick Y. H. LimBiozentrum and the Swiss Nanoscience Institute, University ofBasel, Basel, Switzerland [email protected]

Nuclear pore complexes (NPCs) mediate the traffic of diversesignal-specific cargoes between the cytoplasm and nucleusin eukaryotic cells. This involves numerous intrinsically disor-dered proteins known as phenylalanine-glycine nucleoporins(FG Nups) that lie physically tethered inside each NPC.Importantly, the FG Nups facilitate the selectivity and speed

of cargo-carrying transport receptors (karyopherins or Kaps,specifically importins and exportins) that traverse the NPC.Otherwise, the FG Nups are thought to comprise a barrier thathinders large nonspecific molecules from entering the pore.Still after two decades of research, the NPC modus operandiremains unclear because the FG Nups have eluded direct struc-tural visualization inside the pore. Also, little is known as tohow multivalent Kap-FG Nup interactions promote rapid NPCtranslocation. In my talk, I will highlight our efforts to unravelthe emergent physical principles that underlie such remarkablebiological function. Unexpectedly, our results show that the FGNups are necessary but insufficient for establishing the NPCbarrier. Rather, a surprising finding is that Kaps are essential forboth NPC barrier and transport function. In consolidating theseresults, I will discuss how Kaps might exert control over NPCfunction - as opposed to the general view that NPCs exert con-trol over the transport of Kaps.

Funding

This work is supported by the Swiss National Science Foundation.

References

Kapinos, L. E., Huang, B., Rencurel, C., & Lim, R. Y. H. (2017). Karyopherinsregulate nuclear pore complex barrier and transport function. TheJournal of Cell Biology, 216(11), 3609–3624.

Sakiyama, Y., Mazur, A., Kapinos, L. E., & Lim, R. Y. H. (2016).Spatiotemporal dynamics of the nuclear pore complex transport bar-rier resolved by high-speed atomic force microscopy. NatureNanotechnology, 11(8), 719–723.

Schleicher, K. D., Dettmer, S. L., Kapinos, L. E., Pagliara, S., Keyser, U. F.,Jeney, S., & Lim, R. Y. H. (2014). Selective transport control on molecu-lar velcro made from intrinsically disordered proteins. NatureNanotechnology, 9(7), 525–530.

Schoch, R. L., Kapinos, L. E., & Lim, R. Y. H. (2012). Nuclear transport recep-tor binding avidity triggers a self-healing collapse transition in FG-nucleoporin molecular brushes. Proceedings of the National Academy ofSciences of the United States of America, 109(42), 16911–16916.

73. A ‘double tale’ of evolutionaryaccretion in the structure ofbiological networks

Gustavo Caetano-Anoll"es, Fizza Mughal and M.Fayez AzizDepartment of Crop Sciences, Illinois Informatics Institute,University of Illinois, Urbana, IL 61801, USA [email protected]

The evolution of structure in biology is driven by accretionand change (Caetano-Anoll"es, Caetano-Anoll"es, & Caetano-Anoll"es, 2018). Accretion brings together disparate parts toform bigger wholes. Change provides opportunities forgrowth and innovation. Networks describe how parts associ-ate with each other to form integrated systems. Here weexplain the structure of biological networks with a biphasic(bow-tie) theory of module creation embodied in a ‘doubletale’ of evolutionary accretion (Mittenthal, Caetano-Anoll"es, &Caetano-Anoll"es, 2012). In a first phase, parts are weakly


linked and associate variously. As they diversify, they com-pete with each other and are selected for performance. Theemerging interactions constrain their structure and associa-tions. This causes parts to self-organize into modules withtight linkage. In a second phase, variants of the modulesevolve and become new parts for a new generative cycle ofhigher-level organization. The paradigm predicts the rise ofhierarchical modularity in evolving networks at differenttimescales and complexity levels, which we confirm withphylogenomic and molecular simulation data. Analyses ofevolving networks describing the emergence of metabolism,the rise and diversification of the proteome, the evolution ofthe ribosome, and nanosecond-level change in protein loopdynamics consistently reveal an increase of hierarchicalmodularity and scale-free behavior as networks unfold inevolutionary time (Figure 1). As expected, evolutionary con-straints on network structure are stronger at lower levels ofbiological organization. Remarkably, the phylogenomic data-driven ‘double tale’ of evolutionary accretion was alreadyrecounted in P. Strasb. Gr. Inv. 1665-6, a " 2,000-year-oldpapyrus roll from the ancient city of Panopolis in UpperEgypt attributed to Empedocles and archived at Strasbourg’sNational University Library (Janko, 2004).

Funding

This research has been supported by USDA NIFA award H-1014249 and several Blue Waters supercomputer allocations.

References

Caetano-Anoll"es, G., Caetano-Anoll"es, K., & Caetano-Anoll"es, D. (2018).Evolution of macromolecular structure: A ‘double tale’ of biologicalaccretion and diversification. Science Progress, 101(4), 360–383.

Janko, R. (2004). Empedocles, On nature I 233–364: A new reconstruction ofP. Strasb. Gr. Inv. 1665–6. Zeitschrift Papyrologie Epigraphik, 150, 1–26.

Mittenthal, J. E., Caetano-Anoll"es, D., & Caetano-Anoll"es, G. (2012).Biphasic patterns of diversification and the emergence of modules.Frontiers in Genetics, 3, 147

74. Membranes and machine learning:optimizing the transport of signalsand drugs across membranes

Rachael Mansbacha, Cesar A L"opeza, PaoloRuggeroneb, Bridget S Wilsonc, Nick Hengartnera,Helen I Zgurskayad, Boian Alexandrova and S.Gnanakarana

aTheoretical Division, Los Alamos National Laboratory, Los Alamos,NM; bDepartment of Physics, University of Cagliari, Cagliari, Italy;cDepartment of Pathology and Cancer Center, University of NewMexico, Albuquerque, NM; dDepartment of Chemistry andBiochemistry, University of Oklahoma, Norman, OK

[email protected]

I will discuss two examples where we have used machinelearning on molecular dynamics simulations and experimen-tal data to address the complexity of cell membranes thatpresents a formidable challenge to conventional biophys-ical approaches.

Recently, we suggested a previously unknown molecularmechanism for T-cell activation based on the preferentialbinding and localization of the intrinsically disordered cyto-plasmic signaling tails, which are easily modified by the pres-ence of different lipid species and by the physical state ofthe membrane. Moreover, in a biological membrane withliquid-ordered (Lo) and liquid-disordered (Ld) domains, weshow that specific lipids may play a significant role in immu-noreceptor signaling and similar mechanisms may be pos-sible in a broader context across other signaling pathwayssuch as RAS. We introduce an unsupervised machine algo-rithm based on the non-negative matrix factorization com-bined with custom clustering for analysis of MD simulations.Specifically, we implement this algorithm to detect anddescribe the lateral lipid segregation in a biological mem-brane mimic, a ternary lipid mixture with Lo andLd domains.

The widespread emergence of multi-drug resistance isone of the most serious barriers to effective treatment ofbacterial infections in both public health and biothreatscenarios. Gram-negative bacteria in particular representunique challenges for antibiotic design due to the com-bined effects of their low permeability outer membranesand their nonspecific efflux pumps. Specifically, we haveshown that the low permeability of the two-membrane cellenvelope in P. aeruginosa and the insufficient chemicaldiversity of potential antibiotic compounds present a chal-lenge to antibiotic discovery. We employ both traditionalmachine learning techniques and a novel, fragment-basedapproach to identify the key descriptors and molecular frag-ments within a compound governing permeability andavoidance of efflux, as well as their combined effects in thewild type. This approach allows high-throughput screeningof functionally relevant fragments and offers an alternativeway to search the chemical space for novel anti-biotic candidates.

Figure 1. Log-log plots of the clustering coefficient C(k) as a function of the number of links k for enzyme and subnetwork projections of metabolic enzyme-sub-network bipartite networks as they grow in evolutionary time, billions of years ago (Gya). The scaling is the hallmark of hierarchical modularity. It increases in evo-lution and is stronger at lower levels of metabolic organization.


75. Feature selection inbiomolecular models

Julie MitchellBiosciences Division, Oak Ridge National Laboratory, Oak Ridge,TN, USA [email protected]

Protein–protein interactions regulate many essential bio-logical processes and play an important role in health anddisease. The process of experimentally characterizing proteinresidues that contribute the most to protein–protein inter-action affinity and specificity is laborious. Thus, developingmodels that accurately characterize hotspots at protein–pro-tein interfaces provides important information about how todrug therapeutically relevant protein–protein interactions. Inthis work, we combined the KFC2a protein–protein inter-action hotspot prediction features with Rosetta scoring func-tion terms and interface filter metrics. A 2-way and 3-wayforward selection strategy was employed to train supportvector machine classifiers, as was a reverse feature elimin-ation strategy. From these results, we identified subsets ofKFC2a and Rosetta combined features that show improvedperformance over KFC2a features alone. The forward selec-tion algorithm also helped elucidate the biophysical princi-ples that determine whether a given amino acid is a bindinghot spot.

76. Ultra-fast modeling ofpeptide–MHC interactions with3D convolutional neural net

Mikhail Ignatov, Evangelos Coutsias andDima KozakovLaufer Center for Physical and Quantitative Biology, Stony BrookUniversity, Stony Brook, NY, USA

Association of a peptide with Major HistocompatibilityComplex (MHC) is crucial for adaptive immune system in ver-tebrates. Upon binding to an MHC molecule, the peptide ispresented to T-cells, which triggers an immune response, if

the peptide is recognized as foreign. In recent years person-alized treatment approaches such as cancer immunotherapybased on the knowledge of MHC selectivity of a particularpatient started to emerge. Detection of peptides bound toMHC on the tumor cell surface, containing cancer drivermutations (neoantigens) is essential for efficiency of thetreatment, and therefore, it is important to understandmechanisms, which drive MHC-peptide complex formation.Over the years many complexes have been crystallized andseveral approaches were developed for bound peptide struc-ture prediction. However, existing docking methods are notsuitable for a large scale structural analysis of multiple pepti-des (" 104) due to large execution times, thus faster andmore efficient modeling techniques are required. MachineLearning approaches have demonstrated promising results inprotein structure and ligand binding prediction. Here we pre-sent an ultra-fast peptide-MHC docking method based on3D Convolutional Neural Network (CNN) scoring and con-strained inverse kinematics peptide sampling. Our algorithmis suitable for docking of multiple peptide-MHC complexesand can provide insights for selectivity and preferential bind-ing of different MHC alleles and facilitate structure basedbinding analysis (Figure 1).

77. A revisiting of the RESP chargederivation model

Pavel Ban"a#sa,b, Michal Jane#ceka, Petra K€uhrov"aa,Michal Otyepkaa,b and Ji#r"ı #Sponera,baRegional Centre of Advanced Technologies and Materials,Department of Physical Chemistry, Faculty of Science, Palack"yUniversity, t#r. 17 listopadu 12, Olomouc, 771 46, Czech Republic;bInstitute of Biophysics of the Czech Academy of Sciences,Kr"alovopolsk"a 135, Brno, 612 65, Czech Republic

[email protected]

The representation of the electrostatic interactions byCoulombic interactions between the atom-centered partialcharges is a fundamental part of the molecular mechanicsand empirical force field methods. The broad success of theAMBER force field family originates mainly in the RESPcharge model (Bayly, Cieplak, Cornell, & Kollman, 1993) that

Figure 1.


derives the partial charges to reproduce the electrostaticfield around the molecules.

In this study we revisited the RESP charge derivationmodel in order to improve its description of the electro-static potential around the molecules and thus the descrip-tion of the electrostatic interactions in the force field. Inparticular, we re-optimized the atomic radii used for defin-ition of the grid points for evaluation of the electrostaticfield around the molecule. Some grid points introduced bythe standard RESP procedure using Singh-Merz-Kollmanradii (Singh and Kollman, 1984) are deeply buried into theelectron density, especially in case of aromatic moleculessuch as nucleobases, so that they significantly bias the fit-ted charges in an artificial way. In addition, we redefinedthe restraining scheme, in which we replaced the hyper-bolic restraints toward zero charge values introduced byBayly et al. by weighted parabolic restraints toward CM5charges (Marenich, Jerome, Cramer, & Truhlar, 2012). Theserestraints are able to selectively eliminate the poor statis-tical of the ESP charges of the atoms buried in the mol-ecule. On the other hand, they are negligibly smallwhenever the ESP charges are statistically well-defined. Theredefined charges are able to reproduce the electrostaticpotential around the molecules more accurately thanstandard RESP charges, especially for aromatic systems,and are able, e.g., to entirely eliminate underestimation ofthe base pairing interactions between nucleobases (Banaset al., 2012), which precludes accurate description of thenucleic acids by empirical force fields.

Funding

The authors gratefully acknowledge the support by theMinistry of Education, Youth and Sports of the CzechRepublic and Czech Science Foundation 18-25349S and bythe Operational Programme Research, Development andEducation—European Regional Development Fund, projectno. CZ.02.1.01/0.0/0.0/16_019/0000754.

References

Banas, P., Mladek, A., Otyepka, M., Zgarbova, M., Jurecka, P., Svozil,D., … Sponer, J. (2012). Can we accurately describe the struc-ture of adenine tracts in B-DNA? Reference quantum-chemicalcomputations reveal overstabilization of stacking by molecularmechanics. Journal of Chemical Theory and Computation, 8(7),2448–2460.

Bayly, C. I., Cieplak, P., Cornell, W. D., & Kollman, P. A. (1993). A well-behaved electrostatic potential based method using charge restraintsfor deriving atomic charges: The RESP model. The Journal of PhysicalChemistry, 97(40), 10269–10280.

Marenich, A. V., Jerome, S. V., Cramer, C. J., & Truhlar, D. G. (2012).Charge Model 5: An extension of hirshfeld population analysis for theaccurate description of molecular interactions in gaseous and con-densed phases. Journal of Chemical Theory and Computation, 8(2),527–541.

Singh, U. C., & Kollman, P. A. (1984). An approach to computing electro-static charges for molecules. Journal of Computational Chemistry, 5(2),129–145.

78. Evaluation of the current stateof AMBER DNA force fields

Marie Zgarbov"aa, Michal Otyepkaa, Ji#r"ı #Sponera,b andPetr Jure#ckaaaRegional Centre of Advanced Technologies and Materials,Department of Physical Chemistry, Faculty of Science, Palack"yUniversity, t#r. 17 listopadu 12, Olomouc, 771 46, Czech Republic;bInstitute of Biophysics of the Czech Academy of Sciences,Kr"alovopolsk"a 135, Brno, 612 65, Czech Republic

[email protected]

Force field-based modeling of nucleic acids has witnessedincreasing interest in the past few years. Currently, severalforce field modifications for DNA simulations based on ff99parameters are available in the AMBER suite of programs.Knowing how various conformations are described by avail-able empirical force fields is crucial for molecular modelingof biomolecules, including DNA.

Several refinements of torsion potential (glycosidic poten-tials —vOL3, vOL4; backbone potentials —efOL1 and bOL1)have been suggested in our laboratory. All these refinementswere derived using methodology that includes conform-ation-dependent solvation effects and are available in OL15parameter package. These modifications significantlyimproved description of not only the canonical B-DNA struc-ture but also the noncanonical systems, such as Z-DNA andguanine quadruplexes. Here, we report benchmark simula-tions of various representative DNAs as well as a comparisonof several (bsc0, bsc1 and OL15) force fields. Furthermore, acomparative study of all 136 unique tetranucleotide stepsusing MD simulations with our latest OL15 force field will bepresented. We will provide a comprehensive analysis of theeffect of BII substates on all B-DNA helical parameters andshow how to reliably compare values from experimentand simulation.

Funding

The authors gratefully acknowledge the support by theMinistry of Education, Youth and Sports of the CzechRepublic and Czech Science Foundation no. 17-16107S andby the Operational Programme Research, Development andEducation—European Regional Development Fund projectno. CZ.02.1.01/0.0/0.0/16_019/0000754.

References

Zgarbova, M., Sponer, J., Otyepka, M., Cheatham, T. E., III, Galindo-Murillo, R., & Jurecka, P. (2015). Refinement of the sugar-phosphatebackbone torsion beta for AMBER force fields improves the descrip-tion of Z- and B-DNA. Journal of Chemical Theory and Computation,11, 5723–5736.

Zgarbova, M., Jurecka, P., Lankas, F., Cheatham, T. E., III, Sponer, J., &Otyepka, M. (2017). Influence of BII backbone substates on DNATwist: A unified view and comparison of simulation and experimentfor all 136 distinct tetranucleotide sequences. Journal of ChemicalInformation and Modeling, 57, 275–287.


79. Force fields in trouble—whatcould our DNA and RNA potentialsdo better?

Petr Jure#ckaa, Marie Zgarbov"aa, Ji#r"ı #Sponera,b andMichal OtyepkaaaRegional Centre of Advanced Technologies and Materials,Department of Physical Chemistry, Faculty of Science, PalackyUniversity, 17. listopadu 12, Olomouc 77146, Czech Republic;bInstitute of Biophysics of the Czech Academy of Sciences,Kr"alovopolsk"a 135, Brno 612 65, Czech Republic

[email protected]

While canonical A-RNA and B-DNA nucleic acid structuresseem to be modeled well by current AMBER force fields, thedescription of non-canonical structures is less satisfactory.This has far-reaching consequences, such as problems withsimulations of the nucleic acids folding, which are due toinaccurate description of the unfolded state ensemble. In ourlaboratory we focus on the development of dihedral anglemodifications capable of modeling non-canonical DNA andRNA forms. The key distinguishing feature of our potentialsis the inclusion of conformation-dependent solvation effectsthat were neglected in previous dihedral parameterizationefforts. Several modifications now available in the OL15 pack-age (Zgarbova et al., 2015, OL stands for the city ofOlomouc, Czech Republic) have shown improvements inmodeling of Z-DNA, guanine quadruplexes, non-canonicalRNAs as well as canonical A-RNA and B-DNA duplexes.However, current nucleic acid force fields are still far fromperfect and many new problems are emerging. One exampleis the A/B equilibrium in DNA, which is not reproduced byany of the current AMBER force fields (Zgarbova et al., 2018).Another is excessive destabilization of some important natur-ally occurring a/c backbone substates in RNA (Zgarbova,Jurecka, Sponer, & Otyepka, 2017). Although some of theseproblems may be solvable by further dihedral angle refine-ment, it becomes increasingly clear that balance betweenhydrogen bonding, stacking and interaction with water willhave to be tuned to fully explore accuracy potential of thecurrent non-polarizable force fields.

Funding

The authors gratefully acknowledge the support by theCzech Science Foundation, no. 17-16107S and by theOperational Programme Research, Development andEducation—European Regional Development Fund, projectno. CZ.02.1.01/0.0/0.0/16_019/0000754.

References

Zgarbova, M., Sponer, J., Otyepka, M., Cheatham, T. E. I. I., Galindo-Murillo, R., & Jurecka, P. (2015). Refinement of the sugar % phosphatebackbone torsion beta for AMBER force fields improves the descrip-tion of Z- and B-DNA. Journal of Chemical Theory and Computation,11, 5723–5736.

Zgarbova, M., Jurecka, P., Banas, P., Havrila, M., Sponer, J., & Otyepka,M. (2018). A- to B-DNA transition in AMBER force fields and its cou-pling to sugar pucker. The Journal of Physical Chemistry B, 121,2420–2433.

Zgarbova, M., Jurecka, P., Sponer, J., & Otyepka, M. (2017). Noncanonicala/c backbone conformations in RNA and the accuracy of theirdescription by the AMBER force field. Journal of Chemical Theory andComputation, 14, 319–328.

80. Tuning the hydrogen-bondinginteractions of the AMBER RNAforce field

Petra K€uhrov"aa, Vojt#ech Ml"ynsk"yb, Marie Zgarbov"aa,Miroslav Krepla,b, Giovanni Bussic, Robert B. Bestd,Michal Otyepkaa, Ji#r"ı #Sponera,b and Pavel Ban"a#sa,baRegional Centre of Advanced Technologies and Materials,Department of Physical Chemistry, Faculty of Science, Palack"yUniversity, t#r. 17 listopadu 12, Olomouc 771 46, Czech Republic;bInstitute of Biophysics of the Czech Academy of Sciences,Kr"alovopolsk"a 135, Brno 612 65, Czech Republic; cScuolaInternazionale Superiore di Studi Avanzati, SISSA, via Bonomea265, Trieste 34136, Italy; dLaboratory of Chemical Physics, NationalInstitute of Diabetes and Digestive and Kidney Diseases, NationalInstitutes of Health, Bethesda, MD 20892-0520, USA

[email protected]

Molecular dynamics (MD) simulations became a leading toolfor investigation of structural dynamics of nucleic acids.Despite recent efforts to improve the empirical potentials(force fields, ffs), RNA ffs have persisting deficiencies, whichhamper their utilization in quantitatively accurate simula-tions. Previous studies have shown that at least two salientproblems contribute to difficulties in description of free-energy landscapes of small RNA motifs: (i) excessive stabiliza-tion of the unfolded single-stranded RNA ensemble by intra-molecular base-phosphate and sugar-phosphate interactions(Kuhrova et al., 2016) and (ii) destabilization of the nativefolded state by underestimation of stability of base pairing(Sponer et al., 2018). Here, we introduce a general ff term(gHBfix) that can selectively fine-tune non-bonding inter-action terms in RNA ffs, in particular the H-bonds. gHBfixpotential affects the pair-wise interactions between all pos-sible pairs of the specific atom types, while all other interac-tions remain intact, i.e., it is not a structure-based model. Inorder to probe the ability of the gHBfix potential to refinethe ff non-bonded terms, we performed an extensive set offolding simulations of RNA tetranucleotides and tetraloops.Based on these data we propose particular gHBfix parame-ters to modify the AMBER RNA ff. The suggested paramet-rization significantly improves the agreement betweenexperimental data and the simulation conformational ensem-bles, although our current ff version still remains far frombeing flawless. While attempts to tune the RNA ffs by con-ventional reparametrizations of dihedral potentials or non-bonded terms can lead to major undesired side effects as wedemonstrate for some recently published ffs, gHBfix has a


clear promising potential to improve the ff performancewhile avoiding introduction of major new imbalances.

Funding

The authors gratefully acknowledge the support by theMinistry of Education, Youth and Sports of the CzechRepublic and Czech Science Foundation no. 18-25349S andby the Operational Programme Research, Development andEducation—European Regional Development Fund, projectno. CZ.02.1.01/0.0/0.0/16_019/0000754.

References

Kuhrova, P., Best, R. B., Bottaro, S., Bussi, G., Sponer, J., Otyepka, M., &Banas, P. (2016). Computer folding of RNA tetraloops: Identification ofkey force field deficiencies. Journal of Chemical Theory andComputation, 12(9), 4534–4548.

Sponer, J., Bussi, G., Krepl, M., Banas, P., Bottaro, S., Cunha, R. A., …Otyepka, M. (2018). RNA structural dynamics as captured by molecularsimulations: A comprehensive overview. Chemical Reviews, 118(8),4177–4338.

81. Protein folding and dynamicsin and out of the cytoplasm

Martin GruebeleDept of Chemistry, Univ of Illinois, 600 South Mathews Avenue,Urbana, IL, 61801, USA [email protected]

Martin Gruebele, University of Illinois, will describe how thequinary interactions, sticking and crowding affect proteinfolding and protein–protein interactions in the cytoplasm.Trends from conflicting pressures of sticking and crowdingwill be unraveled by comparing in-cell with in vitro experi-ments, and new techniques to examine protein–proteininteraction dynamics and thermodynamics inside cells revealimportant differences to in vitro assays. Systems range frommetabolic enzymes, to loose protein clusters, to thespliceosome.

82. Protein folding energetics andproteostasis: a two-way connection

Evan T. PowersDepartment of Chemistry, Scripps Research, La Jolla, CA, 92037,USA [email protected]

Proteostasis is the condition of a cell or organism havingenough natively folded protein to carry out essential bio-logical functions while suppressing protein misfolding andaggregation to levels below those that would cause tox-icity. Protein folding to the native state can occur spontan-eously, but often does not, with protein instead becomingtrapped in nonfunctional misfolded or aggregated states.

Because of this, all organisms have a proteostasis network:a collection of chaperones and proteases that shepherdprotein to the native state or recover or dispose of proteinthat has misfolded or aggregated. We have used computa-tional models of in vivo protein folding in the presence ofthe proteostasis network to deepen our understanding ofhow organisms maintain proteostasis. Here, we willdescribe the insights that these models have provided intohow protein folding energetics read through to proteosta-sis and also how these models can, in a sense, beinverted, to use perturbations in proteostasis to learnabout protein folding energetics.

Funding

This research has been supported by the NIH (grantno. GM101644).

83. Simulations of biomoleculesin cellular crowded environments

Yuji Sugitaa,b,c, Kento Kasaharab, Hiraku Oshimab,Isseki Yud, Grzegorz Nawrockie, Suyong Rea andMichael Feigb,eaRIKEN Center for Pioneering Research, 2-1 Hirosawa, Wako-shi,Saitama, 351-0198, Japan; bRIKEN Center for Biosystems DynamicsResearch, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan;cRIKEN Center for Computational Science, 2-1 Hirosawa, Wako-shi,Saitama, 351-0198, Japan; dMaebashi Institute of Technology, 2-1Hirosawa, Wako-shi, Saitama, 351-0198, Japan; eMichigan StateUniversity, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan;

[email protected]

The inside of cell is highly crowded with proteins, nucleicacids, ribosomes, metabolites and so on. The high concentra-tion of proteins realizes macromolecular crowding environ-ments, which can affect protein behaviors in cells. The effectof macromolecular crowding was mainly interpreted via theexcluded volume effect, which favors the compact andglobular structures of proteins in the crowded environment.However, recent in-cell NMR spectroscopy and atomisticmolecular dynamics (MD) simulations in explicit solvent(Harada et al. 2012, 2013) have shown the importance ofweak protein–protein interactions on protein stability anddynamics. In the talk, we discuss the effect of macromolecu-lar crowding on protein-metabolite or protein–ligand interac-tions. In the simulations of the all-atom model ofMycoplasma Genitalium (Yu et al., 2016), we observed thatnot only hydrophobic but also hydrophilic metabolites alsostay on the surfaces of proteins longer than in the bulk solu-tion. Nonspecific and weak protein–metabolite interaction islikely important for the metabolite distributions. We alsoinvestigated kinase-inhibitor binding processes in dilute solu-tion and protein crowded environment by all-atom MD simu-lations and observed different binding processes in thosetwo conditions (Figure 1).


References

Harada, R., Sugita, Y., & Feig, M. (2012). Protein crowding affects hydra-tion structure and dynamics. Journal of the American Chemical Society,134(10), 4842–4849.

Harada, R., Tochio, N., Kigawa, T., Sugita, Y., & Feig, M. (2013). Reducednative state stability in crowded cellular environment due to protein–-protein interactions. Journal of the American Chemical Society, 134,3696–3701.

Yu, I., Mori, T., Ando, T., Harada, R., Jung, J., Sugita, Y., & Feig, M. (2016).Biomolecular interactions modulate macromolecular structure anddynamics in atomistic model of a bacterial cytoplasm. eLife, 5,e19274.

84. Understanding protein behaviorin cells

Gary J. PielakDepartment of Chemistry, University of North Carolina, ChapelHill, NC 27514, USA [email protected]

The crowded and complex environment in cells is predictedto affect protein behavior compared to dilute buffer. Somepredictions may not be correct. We have examined crowdingeffects on the stability of proteins and their complexes. I willfocus on equilibrium data acquired in concentrated solutionsof cosolutes and in living Escherichia coli cells. The cosolutesinclude synthetic polymers and their monomers, other pro-teins and lyophilized cytosol. The results show that crowdingaffects stability and binding, but not always as predicted bysimple theory. The differences point to opportunities for the-oretical efforts and simulations.

Funding

Our research is supported by the National ScienceFoundation (MCB 1410854 and CHE 1607359) and the

National Institutes of Health (R01GM127291). GJP thanks theK.C. Wong Education Foundation for travel support.

References

Guseman, A. J., Perez Goncalves, G. M., Speer, S. L., Young, G. B., &Pielak, G. J. (2018). Protein shape modulates crowding effects.Proceedings of the National Academy of Sciences of the United States ofAmerica, 115(43), 10965–10970.

Guseman, A. J., Speer, S. L., Perez Goncalves, G. M., & Pielak, G. J. (2018).Surface charge modulates protein–protein interactions in physiologic-ally relevant environments. Biochemistry, 57(11), 1681–1684.

Smith, A. E., Zhou, L. Z., Gorensek, A. H., Senske, M., & Pielak, G. J. (2016).In-cell thermodynamics and a new role for protein surfaces.Proceedings of the National Academy of Sciences of the United States ofAmerica, 113(7), 1725–1730.

85. Effect of insulin on DNA-Internucleosomal fragmentation andpoly(ADP-ribose)polymerase 1 activityin rat liver nuclei

Anush Asatryan, Karine Matinyan, Irina Artsruni andEmil GevorgyanDepartment of Biophysics, Yerevan State University, Yerevan,0025, Armenia [email protected]

Apoptosis is the main type of physiological cell death whichensures tissue homeostasis by a dynamic balance betweencell proliferation and death. The final and ‘point of no return’phase of apoptosis is DNA internucleosomal fragmentationresulting in effective degradation of the nuclear DNA and itsfurther elimination by phagocytes (Elmore, 2007). Peptidehormones along with various intra- and extracellular signalstightly regulate the onset of apoptosis by modulating tissueand cell-specific responses. Liver is a target organ where

Figure 1. All-atom MD simulation of the cytoplasm of Mycoplasma genitalium (This figure is taken from Yu et al., 2016).


insulin displays antiapoptotic properties. One of the key fac-tors which mediates the cross-talk of various death pathwaysis poly(ADP-ribose)polymerase 1 (PARP 1), the activity ofwhich is precisely regulated by different exogenous andendogenous cell signals. The activation of PARP 1 by DNAstrand breaks and inhibition of the enzyme by ATP, estab-lishes PARP 1 as a molecular link between DNA damage,energy status and chromatin modification in cells [Kim,Zhang, & Kraus, 2005; Hassa & Hottiger, 2008). PARP 1 playsa prominent role in switching different cell death programsby distinct mechanisms: depleting intracellular NADþ andATP, controlling chromatin accessibility and the activities ofvarious apoptotic endonucleases, which are required for nor-mal nuclear degradation. In present study we examinedwhether insulin can modulate the key process of executionphase of apoptosis-internucleosomal DNA fragmentation, inisolated rat liver nuclei after 4 and 24 h during hormoneadministration to rats. The activity of PARP 1 was concomi-tantly investigated. Our results revealed that insulin sup-pressed the intensity of DNA fragmentation twofold after 4 hof injection. Nevertheless, during 24 h insulin markedly stimu-lated DNA internucleosomal fragmentation. However, insulinhad no appreciable effect on PARP 1 activity in rat livernuclei in all examined periods of hormone action. Takinginto consideration, that the activity of PARP 1 depends onability of the enzyme molecules to bind DNA, we supposethat hydrocortisone’s nuclear receptor can modulate PARP 1activity by competing with the enzyme for binding siteson DNA.

References

Elmore, S. (2007). Apoptosis: A review of programmed cell death.Toxicologic Pathology, 35(4), 495–516.

Hassa, P. O., & Hottiger, M. O. (2008). The diverse biological roles ofmammalian PARPs, a small but powerful family of poly-ADP-ribosepolymerases. Frontiers in Bioscience, 13(13), 3046–3082.

Kim, M. Y., Zhang, T., & Kraus, W. L. (2005). Poly(ADP-ribosyl)ation byPARP-1: ‘PAR-laying’ NADþ into a nuclear signal. Genes &Development, 19, 1951–1967.

86. How do DNA binding proteinsinterpret and modulatenucleosome dynamics?

Habil. Vlad CojocaruIn Silico Biolmolecular Structure and Dynamics Group, TheHubrecht Institute for Developmental Biology and Stem CellResearch, Uppsalalaan 8, Utrecht, CT, 3584, The Netherlands

I will present our recent efforts to explore how linker histo-nes and pioneer transcription factors interpret and modulatenucleosome dynamics to bind DNA wrapped around histo-nes. Linker histones (LH) bind to nucleosomes in a 1:1 stoi-chiometry to form chromatosomes and to compactchromatin fibers. The geometry of the chromatosomeremains debated. Different structures of the LH-nucleosomecomplex solved experimentally or proposed from computer

simulations revealed alternative binding modes for the linkerhistone to nucleosomes. From Brownian and moleculardynamics simulations, we proposed that there is an ensem-ble of chromatosome structures. The LH isoform, nucleosomedynamics and DNA sequence are all factors that influencethe LH binding mode to nucleosomes. Whereas LH proteinsrecognize nucleosomes in a sequence independent manner,the pioneer transcription factors (TFs) are a subclass of TFsthat are able to recognize sequence specific binding sites onDNA wrapped in nucleosomes to regulate gene expression.Pioneer TFs were proposed to contribute to chromatin open-ing. Many of these factors are involved in cell identity transi-tions, and therefore they are of special importance forregenerative therapies. For example, the master regulators ofstem cell pluripotency, Oct4 and Sox2 were proposed to bepioneer TFs. The structural basis for the pionner factor-nucleosome binding remains unknown. Moreover, it is stillnot understood if the binding of TFs to nucleosomes has adirect impact on nucleosome dynamics. To characterize thestructural basis for Oct4-nucleosome recognition, we firstperformed molecular dynamics simulations of 3 nucleo-somes: one with an artificial DNA sequence optimized fornucleosome stability and 2 with native DNA sequences con-taining 1 or multiple Oct4 binding sites, respectively. Wefound that the nucleosome with multiple Oct4 binding siteswas the most mobile. Interestingly, the amplitude of breath-ing and twisting motions in the DNA which were observedin all nucleosomes, was increased in the nucleosome withmultiple Oct4 binding sites. Using an approach that com-bines structural modeling, molecular dynamics simulationswith experimental approaches, we built different models ofOct4-nucleosome complexes and show that alternative butnot all possible configurations are stable and compatiblewith the DNA curvature and DNA-histone interactions.

Acknowledgments

The research presented was performed in collaboration with Mehmet€Ozt€urk, Rebecca Wade, Jan Huertas, Caitlin MacCarthy, and Hans R. Sch€oler.

References

Huertas, J., MacCarthy, C., & Sch€oler, H. R. (2019). Do transcription factorsinterpret nucleosome dynamics? In preparation

€Ozt€urk, M. A., Cojocaru, V., & Wade, R. C. (2018a). Towards an ensembleview of the linker histone - nucleosome complex structure: A para-digm shift from one to many. Structure, 28(8), 1050–1057.

€Ozt€urk, M. A., Cojocaru, V., & Wade, R. C. (2018b). Dependence of chro-matosome structure on linker histone sequence and post-translationalmodifications. Biophysical Journal, 114(10), 2363–2375.

€Ozt€urk, M., Pachov, G., Wade, R. C., & Cojocaru, V. (2016). Conformationalselection and dynamic adaptation upon linker histone binding to thenucleosome. Nucleic Acids Research, 44(14), 6599–6613. doi:10.1093/nar/gkw514

87. Hybrid methods to characterizenucleosome structure and dynamicswith high precision

Anna Panchenko


National Center for Biotechnology Information, National Library ofMedicine, National Institutes of Health, Bethesda, MD, 20894, USA

[email protected]

Nucleosomes are basic units of chromatin compaction andhubs in epigenetic signaling pathways. Nucleosomes experi-ence a broad repertoire of alterations that affect theirdynamics and interactions with various binding partners. Togain insights into intrinsic dynamics of the full nucleosome,we performed all-atom microsecond molecular dynamicssimulations of nucleosomes including linker DNA segmentsand full-length histones (Shaytan et al., 2016). Next, wedeveloped several hybrid approaches by combining experi-mental data with molecular modeling and molecular dynam-ics simulations.

First approach, HYDROID, allows interpretation ofDNA–protein interactions by quantifying Hydroxyl RadicalFootprinting data and integrating it with atomistic structuralmodels (Shaytan et al., 2018). We applied HYDROID to char-acterize Saccharomyces cerevisiae centromeric nucleosomeof unknown structure and identified the precise positioningof centromeric DNA sequence (Shaytan et al., 2017). Inanother study we used HYDROID to pinpoint the footprint ofinteraction between the inner kinetochore protein Mif2/CENP-C and centromeric DNA which plays an important rolefor Mif2 recruitment (Xiao et al., 2017).

Our second hybrid approach was applied to describe his-tone octamer distortion preceding DNA entry into nucleo-somes and processive movement of the ATPase motor ofISW2 on nucleosomal DNA. We performed acceleratedmolecular dynamics simulations guided by experimentalchemical crosslinking data to gain an understanding of thepotential perturbations occurring in the nucleosome struc-ture and investigate how nucleosomal DNA can adjust toperturbations in the histone octamer structure (Figure 1).

References

Shaytan, A. K., Armeev, G. A., Goncearenco, A., Zhurkin, V. B., Landsman,D., & Panchenko, A. R. (2016). Coupling between histone conforma-tions and DNA geometry in nucleosomes on a microsecond timescale:Atomistic insights into nucleosome functions. Journal of MolecularBiology, 428(1), 221–237.

Shaytan, A. K., Xiao, H., Armeev, G. A., Gaykalova, D. A., Komarova, G. A.,Wu, C., … Panchenko, A. R. (2018). Structural interpretation ofDNA–protein hydroxyl-radical footprinting experiments with highresolution using HYDROID. Nature Protocols, 13(11), 2535–2556.

Shaytan, A. K., Xiao, H., Armeev, G. A., Wu, C., Landsman, D., &Panchenko, A. R. (2017). Hydroxyl-radical footprinting combined withmolecular modeling identifies unique features of DNA conformationand nucleosome positioning. Nucleic Acids Research, 45(16),9229–9243.

Xiao, H., Wang, F., Wisniewski, J., Shaytan, A. K., Ghirlando, R., FitzGerald,P. C., … Landsman, D., et al (2017). Molecular basis of CENP-C associ-ation with the CENP-A nucleosome at yeast centromeres. Genes &Development, 31(19), 1958–1972.

88. Investigating the structuraldynamics of DNA in nucleosomecore particles

Lois PollackSchool of Applied and Engineering Physics, Cornell University,Ithaca, NY 14853, USA [email protected]

DNA is tightly packaged around nucleosome core particles(NCPs) for efficient storage, yet must remain readily access-ible for processing. Our interest is in developing experimen-tal tools that reveal the global structure(s) of DNA in NCPs,to understand the molecular mechanisms by which release isaffected by factors including histone or DNA sequence varia-tions or the action of partner molecules, such as remodelersor chaperones. To accomplish this goal, we must be able todetect the global structure(s) of the DNA in the presence ofprotein partners. Contrast variation small angle X-ray scatter-ing is an ideal probe of the conformation of the nucleic acidcomponent of a protein-nucleic acid complex (Tokuda, Pabit,& Pollack, 2016). This method can be implemented in eitherstatic (Chen et al., 2014) or time-resolved studies (Chen et al.,2017). As a proof of principle, we studied the salt dependentconformations of DNA in NCPs, in both equilibrium and timeresolved studies. Data were analyzed using an ensembleoptimization method that yields plausible structural ensem-bles present under each different solution condition or at dif-ferent time during a dynamic release experiment. Initially, we

Figure 1.


employed the tightly positioning Widom 601 DNA sequenceand canonical histones (Chen et al., 2014, 2017); we recentlyexpanded our studies to account for DNA sequence usingsequence specific models to create ensembles (Mauneyet al., 2018), histone variants, and finally the addition ofchaperones such as the Chd-1 remodeler (Tokuda et al.,2018).

Funding

LP’s research on NCPs is supported by NIH underR35GM122514.

References

Chen, Y., Tokuda, J. M., Topping, T., Meisburger, S. P., Pabit, S. A., Gloss,L. M., & Pollack, L. (2017). Asymmetric unwrapping of nucleosomalDNA propagates asymmetric opening and dissociation of the histonecore. Proceedings of the National Academy of Sciences of the UnitedStates of America, 114(2), 334–339.

Chen, Y., Tokuda, J. M., Topping, T., Sutton, J. L., Meisburger, S. P., Pabit,S. A., … Pollack, L. et al (2014). Revealing transient structures ofnucleosomes as DNA unwinds. Nucleic Acids Research, 42(13),8767–8776.

Mauney, A. W., Tokuda, J. M., Gloss, L. M., Gonzalez, O., & Pollack, L.(2018). Local DNA sequence controls asymmetry of DNA Unwrappingfrom nucleosome core particles. Biophysical Journal, 115(5), 773–781.

Tokuda, J. M., Pabit, S. A., & Pollack, L. (2016). Protein-DNA and ion-DNAinteractions revealed through contrast variation SAXS. BiophysicalReviews, 1–11.

Tokuda, J. M., Ren, R., Levendosky, R. F., Tay, R. J., Yan, M., Pollack, L., &Bowman, G. D. (2018). The ATPase motor of the Chd1 chromatinremodeler stimulates DNA unwrapping from the nucleosome. NucleicAcids Research, 46(10), 4978–4990.

89. Revealing dynamic chromatininvasion by pioneer transcriptionfactors on the single-molecule scale

Beat Fierz"Ecole Polytechnique F"ed"erale de Lausanne, SB ISIC LCBM CH B3485, Station 6, Lausanne CH-1015, Switzerland

[email protected]

The dynamic organization of the eukaryotic genome intochromatin is integral to genome regulation. Chromatin struc-ture and dynamics, modulated by histone post-translationalmodifications (PTMs) as well as architectural proteins, dictateDNA access for transcription factors and the gene expressionmachinery. While of key importance, the detailed

mechanisms of DNA access regulation by chromatin dynam-ics are however still poorly understood.

We dissect chromatin signaling on the single-moleculesscale, combining chemical biology approaches and mechan-istic biophysics. We recently developed a single-moleculeFRET (smFRET) approach to directly observe the dynamicarchitecture of chemically defined chromatin fibers. We findthat local chromatin organization is based on tetranucleo-some units, which undergo structural fluctuations on themicro- to millisecond timescale. These dynamics are regu-lated by histone PTMs and linked to function. Indeed,internal chromatin dynamics are exploited by transcriptionfactors (TF) to invade chromatin structure. Employing single-molecule fluorescence imaging we could observe how ayeast pioneer TF, Rap1, binds its target promoter in compactchromatin. Importantly, we show that Rap1 binding openschromatin fiber structure by inhibiting nucleosome-nucleo-some contacts. Finally, we reveal that Rap1 collaborates withthe chromatin remodeler RSC to destabilize promoter nucleo-somes, paving the way to form long-lived bound states onnow exposed DNA.

In summary, our results provide a mechanistic view ofhow a pioneer TF gains access and opens chromatin, therebyestablishing an active promoter architecture and controllinggene expression.

90. Modeling and informatics of DNA,nucleosomes and chromatin frombase pairs to genomes

Ran Sun, Zilong Li and Thomas C. BishopCollege of Engineering and Science, Louisiana Tech University,Ruston, LA, 71272, USA [email protected]

Genomics is a sequence-based informatics science and astructure based molecular science. Nucleosomes are the fun-damental building blocks of chromatin, the biomaterial thathouses the genome in all higher organisms. There areapproximately 160 atomic resolution structures of thenucleosome available from the protein data bank.Collectively they explore histone mutations, species varia-tions, binding of drugs and ionic effects but only a fewsequences of DNA. Given a four-letter code (A, C, G, T) thereare on the order of 4147–1088 possible sequences of DNAthat can form a nucleosome. Exhaustive studies are not pos-sible. Here, we introduce G-Dash, a web based genome dash-board, specifically designed to integrate informatics and 3Dmaterial studies of DNA, nucleosomes and chromatin, andTMB-iBIOMES, a database containing over 20 microsecondsof all atom molecular dynamics simulations representingover 500 different realizations of the nucleosome. G-Dashunites Interactive Chromatin Modeling (ICM), the Biodalliancegenome browser and the JSmol molecular viewer to fold anyDNA sequence into atomic or coarse-grained models of DNA,nucleosomes or chromatin. The exchange of data between


informatics and structure is bi-directional so any informaticstrack can inform a molecular structure (e.g., fold by MNaseactivity) and structure features can be displayed as informat-ics tracks in a genome browser (e.g. Roll, Slide, or Twist). OurTMB-iBIOMES database serves as a reference for comparative,on-demand simulations of nucleosomes. Every simulationdeposited in TMB-iBIOMES contains Amber formatted top-ology and coordinate input files, NAMD formatted output,log and trajectory files, and RMSD and DNA helical param-eter data. Closely related simulations in TMB-iBIOMES aregrouped together and a summary analysis is provided. Alldata can be navigated in a file browser or iBIOMES-Lite webbrowser format or downloaded with command line tools. Itdemonstrates that the workflow and simulation protocolsdeveloped are robust, that DNA sequence can affect thestructure and dynamics of nucleosomal DNA at some loca-tions but not others, and that sequence effects on nucleo-some structure and dynamics can be observed in 10s ofnanoseconds. TMB-iBIOMES and G-Dash work together toprovide a novel means for investigating structure–functionrelationships for regions of the genome ranging from basepairs to chromosomes (Figure 1).

Funding

This effort was supported by NIH IDEA grants 5 P20GM103424-15 and 3 P20 GM103424-15S1 and the NSF OIA-1541079 and the Louisiana Board of Regents.

References

TMB-iBIOMES is available at http://dna.engr.latech.edu/" ibiomes/G-Dash is available at http://dna.engr.latech.edu/" gdash/

91. The effect of tannic acid on PARP1 activity and chromatincondensation in rat thymocyte andliver nuclei

Irina G. Artsruni, Anush L. Asatryan, Karine S.Matinyan and Emil S. Gevorgyan

Department of Biophysics, Yerevan State University, Yerevan 0025,Armenia [email protected]

PAR polymer metabolism play significant role in basal bio-logical processes involved in cell death and survival path-ways. PAR polymer turnover is maintained by coordinatedinterplay of enzymes responsible for PAR synthesis and deg-radation, poly(ADP- ribose)polymerase (PARP 1) andpoly(ADP-ribose) glycohydrolase (PARG) correspondinglly.Nowadays, PARP 1 inhibitors entered into clinical treatmentof cancer patients (Blenn, Wyrsch, & Althaus, 2011) and thesearch of less toxic PARP 1 inhibitors is in progress. It wasdocumented, that natural product-tannic acid (TA) modulatespoly(ADP-ribose) polymer (PAR) catabolism in the in vitroexperimental settings (Czeh, Fabian, S€umegi, & Scorrano,2017). However, little is known about the impact of TA onPARP1 activity and chromatin condensation in in vivo experi-mental settings. Herein, we examine whether intraperitoneal(i.p.) injection of TA to rats could influence PARP 1 activity inrat liver and thymocytes nuclei. The second goal of the studywas investigation of the impact of TA on chromatin accessi-bility to Caþ2/Mgþ2-dependent endonuclease, coming fromthe knowledge that PARP 1 determines chromatin condensa-tion. Our data come to show, that i.p. injection of TA to ratssuppressed PARP 1 activity both in thymocyte and livernuclei and led to augmentation of liver chromatin resistanceto DNA internucleosomal fragmentation. This data indicatedon chromatin condensation in liver nuclei after administra-tion of TA to rats. The in vivo treatment of rats with TAaffects PARP 1 activity in thymocyte and liver nuclei in dose-and organ-specific manner. TA-induced condensation of livernuclei chromatin could provide useful information about thetype of cell death involved in TA-induced hepatotoxicity.

Funding

This work was supported by the RA MES State Committee ofScience, in the frames of the research project no. 18T-1F011.

References

Blenn, C., Wyrsch, P., & Althaus, F. R. (2011). The ups and downs of tan-nins as inhibitors of Poly(ADP-Ribose)glycohydrolase. Molecules, 16(2),1854–1877. doi:10.3390/molecules16021854.

Figure 1 (Left) TMB-iBIOMES is an iBIOMES-lite database with over 20ms of trajectory and analysis data for over 500 unique realizations of the nucleosome.Middle: A collection of Molecular Views representing the interactive capabilities of G-Dash. Right: G-dash integrates the informatics capabilities of a GenomeBrowser (top) with atomic and coarse-grained compute engines via a Control Panel (bottom).


Czeh, A. M., Fabian, Z., S€umegi, B., & Scorrano, L. (2017). Poly(adenosinediphosphate-ribose)polymerase as therapeutic target: Lessons learnedfrom inhibitors. Oncotarget 8(30). 50221–50239. www.impactjournals.com/oncotarget

92. The influence of hydrocortizoneand cisplatin co-administration to ratson chromatin olygonucleosomalfragmentation in liver nuclei

Anush Asatryan, Irina Artsruni, Karine Matinyan andEmil GevorgyanDepartment of Biophysics, Yerevan State University, Yerevan,0025, Armenia [email protected]

Accumulation of cisplatin in normal tissues in the course oftreatment of cancer patients is responsible for severe adverseeffects. Thus, curative effect of cisplatin is attenuated due todose-limiting toxicity and growing resistance of cancer cellsto the drug. To prevent cisplatin-induced side effects, forexample inflammation, nausea and vomiting, hydrocortisoneis commonly co-administrated with cisplatin. Cells thatundergo cytotoxic insults committed life or die decisionsdepending on genetic programs, which could be triggeredby modulation of chromatin condensation. From the otherhand, it is widely recognized that biological activities ofhydrocortisone are mediated by hormone receptors, whichbind and locally modify chromatin structure. However, littleis known about possible effect of co-treatment with cisplatinand hydrocortisone on chromatin structure. To studywhether DNA-binding of hydrocortisone receptors can inter-fere with DNA-cisplatin adducts and alter chromatin struc-ture, we employed chromatin structure-dependent assay,using artificially activated intra-nuclear apoptotic Mgþ2 andCaþ2/Mgþ2-dependent endonucleases (Widlak, Peng, Wang,& Garrard, 2000; Matassov, Kagan, Leblanc, Sikorska, &Zakeri, 2004) and DNase 1 accessibility test. Our datacome to show, that co-administration of cisplatin andhydrocortisone to rats modulated liver chromatin DNase 1accessibility in time-dependent manner. Elevated resistanceto DNase 1 in 4 h after treatment of rats with hydrocorti-sone and cisplatin coincided with the rise of liver DNATm, and was accompanied by descending intensity of DNAinternucleosomal fragmentation. This data prompt thathydrocortisone–cisplatin interaction could prevent looseningof chromatin in internucleosomal chromatin regionsinduced by cisplatin treatment and, thus, might modulateits pharmacological outcomes.

References

Matassov, D., Kagan, T., Leblanc, J., Sikorska, M., & Zakeri, Z. (2004).Measurement of apoptosis by DNA fragmentation. Methods inMolecular Biology (Clifton, N.J.), 282, 1–17.

Widlak, P., Peng, L., Wang, X., & Garrard, W. T. (2000). Cleavage preferen-ces of the apoptotic endonuclease DFF40 (Caspase-activated DNaseor Nuclease) on naked DNA and chromatin substrates. Journal ofBiological Chemistry, 275(11), 8226–8232.

93. Topological polymorphismof chromatin fibers

Davood Norouzi and Victor B. ZhurkinLaboratory of Cell Biology, CCR, National Cancer Institute, NIH,Bethesda, MD 20892, USA [email protected]

Using computer simulations, we found two topologically dis-tinct families of the chromatin fiber conformations distin-guished by the linker length, L. The fibers with L ¼ {10n} and{10nþ 5} bp have DNA linking numbers per nucleosomeLk ) –1.5 and –1.0, respectively (Norouzi and Zhurkin, 2015).The fibers with Lk ) –1.5 (T2) were observed earlier, whilethe topoisomer with Lk ) –1.0 (T1) is novel. These predic-tions were confirmed for circular nucleosome arrays with pre-cisely positioned nucleosomes (Nikitina, Norouzi, Grigoryev, &Zhurkin, 2017). We suggest that topological polymorphism ofchromatin fibers may play a role in the process of transcrip-tion, i. e., the {10nþ 5} DNA linkers are likely to producetranscriptionally competent chromatin structures. Thishypothesis is consistent with available data for several eukar-yotes, from yeast to mouse (Norouzi, Katebi, Cui, & Zhurkin,2015; Norouzi and Zhurkin, 2018). Here, we analyzed thedata from a recent genome-wide radio-probing study ofDNA folding in human cells. The technique uses ionizingRadiation-Induced spatially Correlated Cleavage of DNAwith sequencing (RICC-seq) to identify the DNA-DNA con-tacts that are spatially proximal (Risca, Denny, Straight, &Greenleaf, 2017). We show that the novel topoisomer withLk ) –1.0 has to be taken into account to interpret theexperimental data, especially for the transcriptionally activeregions (Figure 1). This is yet another evidence for occur-rence of two distinct fiber topoisomers (Norouzi andZhurkin, 2018). Potentially, our findings may reflect a gen-eral tendency of chromosomal domains with different lev-els of transcription to retain topologically distinct higher-order fiber conformations, T1 and T2.

References

Nikitina, T., Norouzi, D., Grigoryev, S. A., & Zhurkin, V. B. (2017). DNA top-ology in chromatin is defined by nucleosome spacing. ScienceAdvances, 3(10), e1700957

Norouzi, D., & Zhurkin, V. B. (2015). Topological polymorphism of thetwo-start chromatin fiber. Biophysical Journal, 108(10), 2591–2600.

Norouzi, D., & Zhurkin, V. B. (2018). Polymorphic 30-nm chromatin fiberand linking number paradox. Evidence for the Occurrence of TwoDistinct Topoisomers, bioRxiv 478396; doi: http://dx.doi.org/10.1101/478396.

Norouzi, D., Katebi, A., Cui, F., & Zhurkin, V. B. (2015). Topological diver-sity of chromatin fibers: Interplay between nucleosome repeat length,


DNA linking number and the level of transcription. AIMS Biophysics,2(4), 613.

Risca, V. I., Denny, S. K., Straight, A. F., & Greenleaf, W. J. (2017). Variablechromatin structure revealed by in situ spatially correlated DNA cleav-age mapping. Nature, 541(7636), 237–241.

94. Imaging the 3D organization ofthe chromosome and transcriptomein single cells and the cell atlasof tissues

Xiaowei ZhuangHoward Hughes Medical Institute, Harvard University, 12 OxfordStreet, Cambridge, MA 02138, USA

[email protected]

The spatial organization of genome plays an important rolein many essential genome functions from gene regulation togenome replication. However, many gaps remain in ourunderstanding of the three-dimensional (3D) organization ofchromatin in the nucleus, partly because of the lack ofproper imaging tools to directly visualize this organization.We have developed super-resolution imaging and multiplexfluorescent in situ hybridization (FISH) methods that allowtracing of the 3D conformation of chromatin in cell nucleuswith high resolution. In this talk, I will present these methodsand their applications to the studies of chromatin domainand compartment organization in individual cells.

I will also present a single-cell transcriptome imagingmethod, multiplexed error-robust FISH (MERFISH). MERFISHuses error-robust barcoding, combinatorial labeling and

sequential imaging to enable RNA imaging at the tran-scriptomic scale in individual cells. By allowing single-celltranscriptomic analysis in the native context of cells andtissues, MERFISH facilitates the delineation of gene regula-tory networks, the mapping of RNA distributions insidecells, and the mapping of distinct cell types in complextissues. In this presentation, I will also talk about our tech-nology development of MERFISH and its application to cellatlas mapping.

95. The ‘self-stirred’ genome: bulkand surface dynamics of thechromatin globule

Alexandra ZidovskaCenter for Soft Matter Research, Department of Physics, New YorkUniversity, New York City, USA

Chromatin structure and dynamics control all aspects of DNAbiology yet are poorly understood. In interphase, timebetween two cell divisions, chromatin fills the cell nucleus inits minimally condensed polymeric state. Chromatin serves assubstrate to a number of biological processes, e.g., geneexpression and DNA replication, which require it to becomelocally restructured. These are energy-consuming processesgiving rise to nonequilibrium dynamics. Chromatin dynamicshas been traditionally studied by imaging of fluorescentlylabeled nuclear proteins and single DNA-sites, thus focusingonly on a small number of tracer particles. Recently, wedeveloped an approach, displacement correlation

Figure 1 (Left) Comparison of the experimental RICC-seq data (Risca et al., 2017) with theoretical predictions (Norouzi and Zhurkin, 2018). The experimental gen-ome-wide Fragment Length Distribution (FLD) profiles calculated for the transcriptionally active (H3K27ac, top red curve) and repressed (H3K9me3, top blue curve)regions in human genome correspond to the topoisomer T1 (bottom red curve) and T2 (bottom blue curve), respectively. (Center) Optimal folding of T1 and T2 areshown. (Right) The entry and the exit halves of nucleosomes (gyres) in the left stack are colored differently to emphasize distinct spatial organization of DNA in theT1 and T2 topoisomers, which results in different fragmentation patterns.


spectroscopy (DCS) based on time-resolved image correlationanalysis, to map chromatin dynamics simultaneously acrossthe whole nucleus in cultured human cells (Zidovska, Weitz,& Mitchison, 2013). DCS revealed that chromatin movementwas coherent across large regions (4 and 5lm) for severalseconds. Regions of coherent motion extended beyond theboundaries of single-chromosome territories, suggesting elas-tic coupling of motion over length scales much larger thanthose of genes (Zidovska, Weitz, & Mitchison, 2013). Theselarge-scale, coupled motions were ATP-dependent and uni-directional for several seconds. Following these observations,we developed a hydrodynamic theory (Bruinsma, Grosberg,Rabin, & Zidovska, 2014) and a microscopic model (Saintillan,Shelley, & Zidovska, 2018) of active chromatin dynamics.Here, we investigate chromatin interactions with nuclearenvelope and compare the surface dynamics of the chroma-tin globule with its bulk dynamics (Chu, Haley, & Zidovska,2017), which we also explore using naturally present cellularprobes (Caragine, Haley, & Zidovska, 2018).

References

Bruinsma, R., Grosberg, A. Y., Rabin, Y., & Zidovska, A. (2014). Chromatinhydrodynamics. Biophysical Journal, 106(9), 1871–1881.

Caragine, C. M., Haley, S. C., & Zidovska, A. (2018). Surface fluctuationsand coalescence of nucleolar droplets in the human cell nucleus.Physical Review Letters, 121(14), 148101

Chu, F., Haley, S. C., & Zidovska, A. (2017). On the origin of shape fluctu-ations of the cell nucleus. Proceedings of the National Academy ofSciences of the United States of America, 114(39), 10338–10343.

Saintillan, D., Shelley, M. J., & Zidovska, A. (2018). Extensile motor activitydrives coherent motions in a model of interphase chromatin.Proceedings of the National Academy of Sciences of the United States ofAmerica, 115(45), 11442–11447.

Zidovska, A., Weitz, D. A., & Mitchison, T. J. (2013). Micron-scale coher-ence in interphase chromatin dynamics. Proceedings of the NationalAcademy of Sciences of the United States of America, 110(39),15555–15560.

96. Understanding the humangenome in 3D

Leonid MirneyInstitute for Medical Engineering & Science, MassachusettsInstitute of Technology, 77 Massachusetts Ave E25-406,Cambridge, MA 02139, USA [email protected]

Leonid Mirny is co-director of the Center for 3D Structureand Physics of the Genome at UMass Medical School andMIT, funded by the National Institutes of Health’s 4DNucleome Program. He is working to understand the humangenome in 3D with his team at MIT in collaboration with theDekker Lab at UMass Medical School. Using new data uncov-ered via Chromosome Conformation Capture (Hi-C) technol-ogy and computer simulations, the collaborators explorehow the genome is organized inside a cell.

97. Applications of modificationdetection in nanopore sequencing

Winston TimpBiomedical Engineering, Johns Hopkins University, Baltimore, MD,USA [email protected]

Nanopore sequencing is a single molecule characterizationmethod, allowing direct sequencing of DNA or RNAsequencing with read lengths ranging from kilobases toeven megabases. Unlike traditional sequencing-by-synthesismethods, it can distinguish covalently modified nucleotidesdirectly through their modulation of the electrolytic cur-rent. And the long reads allow for straightforward detec-tion of structural variations, large insertions, deletions ortranspositions that are often difficult to detect with short-read sequencing.

We demonstrate the power of this technique, as appliedwith DNA, combined with exogenous labeling, to performan integrative, single-molecule characterization of the epige-nome. We used M.CviPI, a GpC methyltransferase, to labelaccessible chromatin in cancer and normal cell lines. Thisallowed us to simultaneously correlate nucleosome position-ing and native CpG methylation along long (" 10 kb) singlemolecules. We investigated methods of targeted sequencingfor deeper nanopore sequencing at specific genomic loci.These methods resulted in focussed coverage of long nativenanopore sequencing reads, measuring single-moleculemethylation patterns, SVs and SNVs. Finally, we haveapplied native RNA sequencing to interrogate poly(A) taillengths and modifications to RNA which may be involvedin posttranscriptional regulation. RNA base modifications aredetectable via modulation of the nanopore current; our ini-tial focus is on the METTL3 motif (GGm6ACU). Poly-A taillengths inform mRNA lifetime; we can measure theselengths from how long the molecule takes to transit thepore. With these methods, we have measured gene specificand even isoform specific poly(A) tail lengths and modifica-tion signals.

98. Stoichiometric constraints inprotein sequences

Aditya MittalKusuma School of Biological Sciences, IIT Delhi, Hauz Khas, NewDelhi 110016, India [email protected]

About a decade ago, rigorous analyses of structural data ofthousands of naturally occurring folded proteins yielded asurprising ‘margin of life’ for stoichiometric composition, i.e.,percentage occurrence of individual amino acids, of proteinsequences (Mittal et al., 2010; Mittal & Jayaram, 2011a,2011b). This ‘margin of life’ refers to the lower than expected


variances in percentage occurrence of individual amino acidsin protein sequences (Mezei, 2011). The concept of ‘marginof life’ is about a decade old now and has been confirmedover a large sequence space for almost all known proteinsequences (even in the absence of structural data for a largenumber of sequences). While the earlier work was based onthe largest structural dataset at that time, in this work furtherexplorations on compositional constraints for known proteinsequences are presented on the largest dataset analyzed tilldate. The relationships between peptide-bonded pairs andtriplets of amino acids, in about half-a-million proteinsequences, with their various physico-chemical propertiesand individual proportions (of the peptide-bonded pairs andtriplets) are explored. While having profound evolutionaryimplications towards our insights into occurrence of naturallyoccurring protein sequences, the results discussed also prom-ise to serve as guide for creating stable and structurally con-trolled and/or ‘disordered’ designer proteins.

References

Mezei, M. (2011). Discriminatory power of stoichiometry-driven proteinfolding? Journal of Biomolecular Structure and Dynamics, 28(4),625–626.

Mittal, A., Jayaram, B., Shenoy, S. R., & Bawa, T. S. (2010). A stoichiometrydriven universal spatial organization of backbones of folded proteins:Are there Chargaff’s rules for protein folding? Journal of BiomolecularStructure and Dynamics, 28(2), 133–142.

Mittal, A., & Jayaram, B. (2011a). Backbones of folded proteins revealnovel invariant amino acid neighborhoods. Journal of BiomolecularStructure and Dynamics, 28(4), 443–454.

Mittal, A., & Jayaram, B. (2011b). The newest view on protein folding:Stoichiometric and spatial unity in structural and functional diversity.Journal of Biomolecular Structure and Dynamics, 28(4), 669–674.

99. Tongue tumor proteinBAH14511.1 sequence analysis andhomology modeling to find outinteracting binder network throughmolecular docking

Snigdha Singh and Ramesh ChandraDrug Discovery & Development Laboratory, Department ofChemistry, University of Delhi, Delhi 110007, India

[email protected]

Day by day, people are dying of one or the other type ofcancer worldwide; oral cancer being the third most commoncancer in India and sixth in the world. To treat cancer, theroles of different factors involved in triggering cancer needsto be exploited which needs the exploration of mechanismsof the factors involved and targeting their interactions withco-operating factors (Takiar et al., 2010; van der Wall, 2013).The protein product with accession number BAH14511.1, iso-lated from tongue tumor tissue of Homo sapiens was foundto be an isoform b of cGMP-dependent protein kinase II(PKGII) as shown by the results of nonredundant BLAST.PKGII plays an important role in the regulation of signalingpathways associated with cancer, and hence prediction ofthe 3D structure of the isoform would be important forthe prediction of lead molecules that can be further usedfor designing a drug for the regulation of PKGIIb. In thisstudy, various tools (PROTPARAM, SMART, TMHMM, SignalP,SecretomeP, NetChop and NetPhos) were used for func-tional annotation. The 3D structure of the protein waspredicted by Homology Modeling approach (Blom,Gammeltoft, & Brunak, 1999; Yu, Chen, Lu, & Hwang,2006). The 3D structure was validated by Verify3D andWhatIf. The binding site in the 3D structure of PKGIIb waspredicted by using SiteMap which gave 5 sites as resultfrom which the site having highest site score of 1.114 andvolume 348.145 was further used for docking and virtualscreening. The predicted 3D structure was used to screenthe NCI database for finding potential binders. The struc-tures from the NCI database were docked with the 3Dstructure of the protein and the best docking score of% 12.547 was obtained. All the compounds binding withthe protein followed Lipinski’s Rule. The top structuresfrom the NCI database have been reported as potentialbinders of the protein. The binding affinity of the topbinder NSC1972 was found to be % 63.296 as predicted byPRIME MM-GBSA and Induced-fit docking was done to val-idate docking results (Figure 1).

References

Blom, N., Gammeltoft, S., & Brunak, S. (1999). Sequence and structure-based prediction of eukaryotic protein phosphorylation sites. Journalof Molecular Biology, 294(5), 1351–1362.

Takiar, R., Nadayil, D., & Nandakumar, A. (2010). Projections of number ofcancer cases in India (2010-2020) by cancer groups. Asian PacificJournal of Cancer Prevention, 11(4), 1045–1049.

Figure 1.


van der Waal, I. (2013). Are we able to reduce the mortality and morbid-ity of oral cancer; Some considerations. Medicina Oral, Patologia Oraly Cirugia Bucal, 18(1), e33–e37.

Yu, C. S., Chen, Y. C., Lu, C. H., & Hwang, J. K. (2006). Prediction of pro-tein subcellular localization. Proteins: Structure, Function, andBioinformatics, 64(3), 643–651.

100. Can R-loops themselves be themysterious R-lesions?

Elena A. Kouzminova and Andrei KuzminovUniversity of Illinois at Urbana-Champaign, B103 CLSL, 601 SouthGoodwin Avenue, Urbana, IL 61801, USA

[email protected]

All free-living organisms have two kinds of RNase Henzymes (activities that remove the RNA nucleotides fromthe RNA:DNA hybrid duplexes): ‘type I’ attacks R-loops and& 4nt long R-tracts, whereas ‘type II’ attacks 50RNA-DNA3’junctions, mostly at single RNA nucleotides in DNA. Type IImutants (rnhB in E. coli) behave like WT, suggesting thatsingle rNs in DNA are innocuous, while type I mutants(rnhA in E. coli) are inhibited, suggesting that not only R-loops form frequently in the chromosomal DNA, but thatthey are also quite stable and, unless actively hydrolyzed,interfere with replication and transcription. Surprisingly, thecombined type I/II RNase H defect reveals synergy: the dou-ble rnhAB mutant E. coli barely grows and forms long fila-mentous cells that have problems with nucleoidsegregation. We have argued that the chromosomal prob-lems of the double rnhAB mutant reflect formation of R-lesions: RNA-containing DNA lesions that interfere withchromosomal replication and segregation.

Since the only common substrate for the two bacterialRNase H types is R-tract, we propose that the double rnhABmutants accumulate R-tracts in their DNA that eventuallylead to irreparable double-strand breaks. This idea is con-sistent with the following phenotypes of the double rnhABmutants over the single rnhA or rnhB mutants: (1) increasedSOS response; (2) dependence on recombinational repair;(3) formation of double-strand breaks in the chromosomalDNA; (4) sensitivity to inhibition of translation (shouldinduce R-loops); (5) sensitivity to inhibition of chromosomalreplication (should preserve R-loops); (6) RNase-treatment-induced fragmentation of the chromosomal DNA. Theemerging mechanism envisions that in the absence ofRNase H, a fraction of stabilized R-loops is transformed intoR-tracts, that are converted into R-gaps by replication, thelatter being attacked by cellular RNases, yielding double-strand DNA breaks.

However, studying one peculiar phenotype of the rnhABmutants shows how an R-loop could itself be an integralpart of an R-lesion. The rnhAB mutants are extremely UV-sensitive, but at the same time are not generally sensitiveto other forms of DNA damage. We show that in UV-edrnhAB mutants: (1) while no additional double-strand breaksare formed, post-UV replication recovery is slow and

incomplete, suggesting replication fork impediments; (2) itis inhibition of transcription by pyrimidine dimers (PDs) inDNA, rather than inhibition of translation by PDs in mRNA,that is responsible for the sensitivity; (3) promotion/stabil-ization of R-loops exacerbates the sensitivity, while theirdestabilization reduces the sensitivity; (4) PDs are rapidlyremoved from the bulk DNA, yet " 10% of PDs in thechromosome are removed slowly; (5) RNA:DNA hybridsaccumulate massively in genomic DNA after UV. We arecurrently testing whether the chromosomal regions contain-ing UV-induced RNA:DNA hybrids are enriched for slowly-removable PDs. If yes, then UV-induced R-lesions could beRNA polymerases stalled at PDs in template DNA, whileanchored on their 5’-side by R-loops, an arrangement thatshould block PD removal.

Funding

This research is supported by grant GM 073115 from theNational Institutes of Health.

References

Kouzminova, E. A., Kadyrov, F. F., & Kuzminov, A. (2017). RNase HII savesrnhA mutant Escherichia coli from R-loop-associated chromosomalfragmentation. Journal of Molecular Biology, 429(19), 2873–2894.PMID: 28821455

101. Characterization of R loopsin class switch recombination

Jacqueline Barlow and Hongchang ZhaoDepartment of Microbiology and Molecular Genetics, University ofCalifornia Davis, Davis, CA 955616, USA

Recurrent DNA translocations characterize blood cell cancers,and are frequently products of aberrant repair of pro-grammed DNA double-strand breaks (DSBs). Defining theprecise molecular mechanisms governing DSB generationand repair is critical to revealing how lymphoid cancers arise.The majority of cancers involving antibody-producing B cellsarise during class switch recombination (CSR), a programmedDNA repair event at the immunoglobulin heavy chain (IgH)locus. CSR is initiated by DSBs generated by the enzyme AID,whose recruitment to IgH requires transcription. AID-depend-ent DSB formation strongly correlates with the appearanceof R loops—three-stranded nucleotide structures wherenewly transcribed RNA re-anneals to template DNA. ThoughR loops were observed at the IgH locus over 20 years ago,their role in CSR remains undefined. To investigate the role Rloops play in CSR, we generated mice deficient for twoenzymes promoting R loop dissolution, the helicaseSenataxin (SETX-/-) and the nuclease RNase H2 (RNH2Bf/f). Wefind that B cells from SETX-/- RNH2Bf/f mice have increased Rloop formation, and over 10% of cells accumulate unrepairedDNA breaks and translocations at IgH. In contrast, WT andsingle mutants show modest IgH instability and limited


increases in R loop formation. All 4 genotypes are proficientfor CSR, thus DSB repair is largely intact. These results showthat Setnataxin and RNase H2 act independently to promotegenome stability and suppress aberrant DNA repair duringCSR. Sequence analysis revealed an increased frequency ofinsertions and deletions at class switch junctions in double-deficient cells, a mutational signature associated withincreased repair via alternative end-joining. We propose thatSenataxin and Rnase H2 promote classical NHEJ by removingR loops during CSR.

Funding

This research has been supported by an NCI CareerTransition Award, K22CA188106

102. Mechanisms of R-loop-inducedchromosome fragility at structure-forming repeats

Xiaofeng Allen Su, Simran Kaushal, Ruby Ye andCatherine H. FreudenreichDepartment of Biology, Tufts University, Medford, MA 02155, USA

[email protected]

CAG repeats are structure-forming repetitive DNA sequences,and expansion beyond a threshold of " 35 CAG repeats isthe cause of many human diseases including Huntington’sdisease and myotonic dystrophy. Expanded CAG repeats areprone to breakage, and repair of the breaks can cause repeatcontractions and expansions. We utilized an expanded CAG-70 repeat inserted into a yeast artificial chromosome (YAC)system to evaluate mechanisms of repeat fragility andinstability (contractions and expansions). Upon deletion ofyeast RNase H genes, RNH1 and RNH201, we found an ele-vated level of RNA:DNA hybrids at CAG-70 repeats in vivo byusing chromatin immunoprecipitation (ChIP). Through CAGrepeat fragility analysis, we discovered that CAG-70 repeatsshow a dramatic and significant increase in fragility in theabsence of RNase H genes compared to wild-type, indicatinghigher levels of breakage events at the expanded repeatsduring R-loop formation (Su & Freudenreich, 2017). In con-trast, fragility of another structure-forming repeat composedof an AT dinucleotide was not affected by RNase deletion oroverexpression. Significant increases in CAG-70 repeatinstability were also observed in the RNase H deletion back-ground that were dependent on the base excision repairpathway (Su & Freudenreich, 2017). Since disease-causingCAG repeat expansions occur in transcribed regions shownto harbor R-loops, our findings indicate that R-loop-mediatedfragility is a mechanism that could cause DNA damage andrepeat-length changes in human cells.

In a search for the cause of R-loop-mediated fragility, wefound that a yeast cytosine deaminase, Fcy1, was enriched atthe expanded CAG repeats in rnh1Drnh201D cells. Deletionof Fcy1 significantly decreases CAG-70 repeat fragility in thernh1Drnh201D background, and reduces the elevated CAG

repeat contractions back to the wild-type level (Su &Freudenreich, 2017). These findings provide evidence for anovel mechanism for CAG repeat fragility mediated by cyto-sine deamination of DNA engaged in R-loops. In addition, asecond mechanism of R-loop-mediated fragility at CAGrepeats was identified to be dependent on the Mlh1-Mlh3nuclease. This result could explain the previously demon-strated role of Mlh1-Mlh3 in causing CAG repeat expansionsin a mouse model (Pinto et al., 2013). Thus, R-loop-medi-ated cleavage could be an important determinant ofinstability at unstable and expandable repeats in the humangenome.

References

Pinto, R. M., Dragileva, E., Kirby, A., Lloret, A., Lopez, E., St Claire, J., …Wheeler, V. C. (2013). Mismatch repair genes Mlh1 and Mlh3 modifyCAG instability in Huntington’s disease mice: Genome-wide and can-didate approaches. PLoS Genetics, 9(10), e1003930

Su, X. A., & Freudenreich, C. H. (2017). Cytosine deamination and baseexcision repair cause R-loop-induced CAG repeat fragility andinstability in Saccharomyces cerevisiae. Proceedings of the NationalAcademy of Sciences of the United States of America, 114(40),E8392–E8401.

103. Understanding and targetingR loops in cancer cells

Lee Zoua,baMassachusetts General Hospital Cancer Center, Boston, MA, USA;bDepartment of Pathology, Harvard Medical School, Boston, MA,USA [email protected]

R loops arising during transcription-induced genomicinstability, but how cells respond to the R loop-associatedgenomic stress is still poorly understood. Our recent studieshave suggested that ATR is involved in the sensing of Rloops. Here, we show that cells harboring high levels of Rloops rely on the ATR kinase for survival. In response toaberrant R loop accumulation, the ATR-Chk1 pathway isactivated by R loop-induced reversed replication forks. ATRprotects the genome from R loops by suppressing transcrip-tion-replication collisions, promoting replication fork recov-ery, and enforcing a G2/M cell-cycle arrest. Furthermore,ATR prevents excessive cleavage of reversed forks byMUS81. These results suggest that ATR is a key sensor andsuppressor of R loop-induced genomic instability, uncover-ing a signaling circuitry that safeguards the genome againstR loops.

We also find that ATR is important for survival of cancercells harboring high levels of R loops. Heterozygous somaticmutations in spliceosome genes (U2AF1, SF3B1, ZRSR2 orSRSF2) occur in >50% of myelodysplastic syndrome (MDS)and AML patients. We show that RNA splicing perturbationby expression of the U2AF1(S34F) mutant causes accumula-tion of R loops and elicits an ATR response. ATR inhibitors(ATRi)-induced DNA damage and cell death in U2AF1S34F-


expressing cells, and these effects of ATRi were enhanced bysplicing modulating compounds. Our results suggest thatATR may represent a novel therapeutic target in MDSpatients carrying the U2AF1(S34F) mutation and potentiallyother malignancies harboring spliceosome mutations.

104. The role of the redox active4Fe–4S cluster of yeast Dna2

Siobh"an Gaustad MacArdle and Jacqueline BartonDepartment of Chemistry and Chemical Engineering, CaliforniaInstitute of Technology, 1200 E. California Blvd., MC 127-72,Pasadena, CA, 91126, USA [email protected]

Dna2 is an essential nuclease-helicase conserved throughouteukaryotic organisms with an impressive repertoire of DNAmaintenance activity (Budd & Campbell, 1995). Due to itsmajor roles in Okazaki fragment processing during DNA repli-cation, double strand break repair, telomere maintenanceand mitochondrial DNA maintenance, it is not surprising thatboth Dna2 upregulation and mutation have been observedin various types of cancer (Lee, Kim, Yoo, & Lee, 2010;Strauss, 2014). Despite the fact that both the helicase andnuclease domains are equally conserved in evolution, thenuclease function of Dna2 dominates on most substratesand the functional switch between helicase and nucleaseactivity is regulated by an unclear mechanism, which mayinvolve the 4Fe–4S cluster (Pinto, Kasaciunaite, Seidel, &Cejka, 2016). The goal of this work is to elucidate the effectof the redox properties of the 4Fe–4S cluster on the regula-tion of helicase and nuclease activity in S. cerevisiae Dna2.We have overexpressed wild type S. cerevisiae Dna2 andnuclease-dead Dna2 E675A in E. coli and purified the recom-binant enzymes with sufficient yield and purity. Using multi-plexed DNA-modified Au electrodes, we have characterizedthe DNA-bound electron transfer activity of isolated Dna2.Cyclic voltammetry on DNA-modified electrodes showedreversible DNA-bound redox activity centered at " 90mV vs.NHE. This redox potential is similar to that of other DNA-binding 4Fe–4S enzymes that we have studied and supportsa DNA-mediated redox signaling role for the 4Fe–4S cofactorin regulating Dna2 activity. The redox potential of Dna2unbound to DNA has been determined by protein film elec-trochemistry to be " 150mV vs. NHE, 60mV greater thanthat of the DNA-bound protein. This positive shift in redoxpotential suggests that DNA binding activates the 4Fe–4Scluster towards oxidation and is consistent with what hasbeen observed for other DNA-binding proteins containing a4Fe–4S cluster (Bartels et al., 2017). Bulk oxidation andreduction of Dna2 on DNA-modified electrodes has beenperformed revealing an increase in signal after oxidationand decrease upon reduction, consistent with an increasein DNA-binding affinity upon oxidation of the 4Fe–4S clus-ter to the 3þ state. This study illuminates the biochemicalrole played by the 4Fe–4S cluster and provides insight into

the mechanism through which Dna2 promotes gen-ome stability.

References

Bartels, P. L., Zhou, A., Arnold, A. R., Nu~nez, N. N., Crespilho, F. N.,David, S. S., & Barton, J. K. (2017). Electrochemistry of the [4Fe4S]cluster in base excision repair proteins: Tuning the redox potentialwith DNA. Langmuir: The ACS Journal of Surfaces and Colloids, 33(10),2523–2530.

Budd, M. E., & Campbell, J. L. (1995). A yeast gene required for DNA rep-lication encodes a protein with homology to DNA helicases.Proceedings of the National Academy of Sciences of the United States ofAmerica, 92(17), 7642–7646.

Lee, S. H., Kim, Y. R., Yoo, N. J., & Lee, S. H. (2010). Mutation and expres-sion of DNA2 gene in gastric and colorectal Carcinomas. KoreanJournal of Pathology, 44(4), 354–359.

Pinto, C., Kasaciunaite, K., Seidel, R., & Cejka, P. (2016). Human DNA2possesses a cryptic DNA unwinding activity that functionally integra-tes with BLM or WRN helicases. eLife, 5

Strauss, C. (2014). The DNA2 nuclease/helicase is an estrogen-dependentgene mutated in breast and ovarian cancers. Oncotarget, 5,9396–9409.

105. Primer handoff between DNAprimase and DNA polymerase a

Aoshu Zhonga, Lauren E. Salayb, Walter J. Chazinb

and Jacqueline K. BartonaaDivision of Chemistry and Chemical Engineering, CaliforniaInstitute of Technology, Pasadena, CA 91125, USA; bDepartmentsof Biochemistry and Chemistry, Center for Structural Biology,Vanderbilt University, Nashville, TN 37240, USA

[email protected]

DNA replication is a fundamental process. In eukaryotes, thefirst step in daughter strand synthesis is the generation of ashort (" 10 nt) RNA primer by DNA primase, followed bytransfer of the primer to DNA polymerase a (Pol a) for exten-sion by " 20 nts (O’Brien et al., 2017; Burgers & Kunkel, 2017;Pellegrini, 2012; O’Brien et al., 2018; O’Brien, Holt, Salay,Chazin, & Barton, 2018). Although the catalytic activity andstructures of primase and Pol a have been extensivelystudied (Burgers & Kunkel, 2017; Pellegrini, 2012; O’Brienet al., 2018; O’Brien, Holt, Salay, Chazin, & Barton, 2018), themolecular mechanism of primer handoff between primaseand Pol a remains largely unknown. We have proposed thatredox switching of the [4Fe4S] cluster in primase is import-ant for primer truncation and handoff to Pol a (O’Brien et al.,2017; O’Brien et al., 2018). Eukaryotic Pol a is composed of acatalytic subunit (p180) and a regulatory subunit (p68). Thep180 subunit contains a [4Fe4S] cluster. Thus, we hypothe-size that the redox partner of primase is Pol a and that thetwo [4Fe4S] clusters orchestrate the primer handoff betweenthe two [4Fe4S] proteins using DNA-mediated charge trans-port chemistry. Here, we present results using anaerobicallypurified human [4Fe4S] (C-terminal domain of p180 (p180C)in complex with p68. To the best of our knowledge, this isthe first time that p180C-p68 is isolated with an [4Fe4S]


cluster bound. The cluster loading can be improved to nearly100% after initial purification using a reconstitution protocol.The protein exhibits electrochemical properties similar to thep58C [4Fe4S] domain of primase. Using multiplexed DNA-modified Au electrodes, we observe a large cathodic peakbetween –170 and –180mV versus NHE by cyclic voltamme-try only during the initial scan to negative potentials afteroxidation by bulk electrolysis. As with p58C, the data areconsistent with a model where p180C-p68 is more tightlyassociated with DNA in the oxidized state compared to thereduced state. We also present results from primer elong-ation assays, where truncated products are formed to agreater extent in the presence of the primase-p180C-p68complex versus primase alone. When electrochemically oxi-dized p180C-p68 was assayed together with primase, thelength of the products is similar to the products producedwith primase alone. Together, these data are consistent withour hypothesis that the [4Fe4S] clusters in p180C-p68 andprimase regulate the primer handoff from primase to Pol a.

References

Burgers, P. M. J., & Kunkel, T. A. (2017). Eukaryotic DNA replication fork.Annual Review of Biochemistry, 86, 417–438.

O’Brien, E., Holt, M. E., Thompson, M. K., Salay, L. E., Ehlinger, A. C.,Chazin, W. J., & Barton, J. K. (2017). The [4Fe4S] cluster of humanDNA primase functions as a redox switch using DNA charge transport.Science, 355, eaag1789.

O’Brien, E., Salay, L. E., Epum, E. A., Friedman, K. L., Chazin, W. J., &Barton, J. K. (2018). Yeast require redox switching in DNA primase.Proceedings of the National Academy of Sciences of the United States ofAmerica, 115(52), 13186–13191.

O’Brien, E., Holt, M. E., Salay, L. E., Chazin, W. J., & Barton, J. K. (2018).Substrate binding regulates redox signaling in human DNA primase.Journal of the American Chemical Society, 140, 17153–17162.

Pellegrini, L. (2012). The Pol a-primase complex. Sub-CellularBiochemistry, 62, 157–169.

106. Annotation of cysteine functionsin unknown proteins—a newapproach based on proteinmicroenvironments

Debashree Bandyopadhyay, Akshay Bhatnagar andShubham PaliwalBirla Institute of Technology and Science, Pilani, HyderabadCampus, Hyderabad 500078, India

[email protected]

Huge disparity exists in terms of number of sequenced pro-teins, experimentally available protein structures and func-tionally characterized proteins. With the advent of proteomictechniques large number of structures are solved for‘proteins of unknown functions’ (PUFs). More than 20% ofthe known protein domains are characterized as ‘domains ofunknown function’ (DUFs). Bacteria and eukaryotes sharenear about 1000 DUFs. Evolutionary conservation suggeststhat DUFs are essential in both eukaryotes and bacterialpathogens. However, the function of those proteins andinvolvement of important amino acids in protein functionsremain elusive. Characterization of functions even for a fewproteins or domains is experimentally laborious and timeconsuming. Here, we present annotation of cysteine aminoacid functions based on protein microenvironments inknown protein dataset and apply those annotations tounknown proteins. Protein microenvironment is described bytwo parameters, buried fraction (BF) and rHpy (a quantitativeproperty descriptor to describe the hydrophobicity of localenvironment around an amino acid) (Bandyopadhyay &Mehler, 2008). Cysteine functions were curated from litera-ture and structures were obtained PDB database with reso-lution greater than 1.5 Å. Statistically meaningfulcharacterization was done for four cysteine modifications,namely disulfide formation (Bhatnagar, Apostol, &Bandyopadhyay, 2016), thioether formation, sulfenylationand metal binding (Bhatnagar & Bandyopadhyay, 2018).Distinct contours (within the space of BF and rHpy) wereidentified for different cysteine modifications. Training setson four cysteine functions were validated on test sets ofcysteines obtained from protein structures in PDB databasewith resolution lower than 1.5 Å. These contours will beexploited to annotate cysteine functions in PUFs and DUFs(Figure 1).

Funding

This research is supported by Funding from Department ofScience and Technology (DST-SERB), Govt. of India (File No.EMR/2017/002953)

References

Bandyopadhyay, D., & Mehler, E. L. (2008). Quantitative expression ofprotein heterogeneity: Response of amino acid side chains to theirlocal environment. Proteins: Structure, Function and Bioinformatics,72(2), 646–659.

Figure 1.


Bhatnagar, A., Apostol, M. I., & Bandyopadhyay, D. (2016). Amino acidfunction relates to its embedded protein microenvironment: A studyon disulphide-bridged cystine. Proteins: Structure, Function, andBioinformatics, 84(11), 1576–1589.

Bhatnagar, A., & Bandyopadhyay, D. (2018). Characterization of cysteinethiol modifications based on protein microenvironments and localsecondary structures. Proteins: Structure, Function, and Bioinformatics,86(2), 192–209.

107. Change in the yeastPma1Hþ-ATPase regulation causesredistribution ofpolyphosphates (PolyP)

Alexandr A. Tomashevsky and Valery V. PetrovSkryabin Institute of Biochemistry and Physiology ofMicroorganisms, Russian Academy of Sciences, Pushchino, 142290,Russia [email protected]

Plasma membrane Pma1Hþ-ff!#ase is a key enzyme ofyeast metabolism. The enzyme functioning is regulated byglucose, which fermentation leads to producing ATP beingused for phosphorylation of proteins and other substratesand maintaining energy metabolism. Glucose-induced activa-tion of Pma1 is structurally accompanied by the enzymemultiple and reversible phosphorylation during intracellulartraffic via secretory pathway on route to plasma membrane.There is evidence that the Pma1 activation is linked to Ser/Thr phosphorylation within the regulatory C-terminus and itleads to essential conformational changes; S911 and T912were identified as two major phosphosites, which phosphor-ylation goes tandemly (Lecchi et al., 2007). The location ofall sites of phosphorylation is still unclear; however, it seemslogical that phosphorylation of Pma1 goes step by step andsuch sites could be located both in the enzyme intra- andextracellular parts. Both ATP and PolyP can phosphorylateSer and Thr like in the case of Lys (Azevedo et al., 2015); wehave earlier shown that Ala replacement of phosphorylableresidues in the extracellular L9-10 loop, which is close to theC-tail, has affected PolyP distribution (Tomashevsky & Petrov,2011, 2013). Other extracellular phosphorylable residueswere found not to be important for the enzyme structure-function relationship (Petrov, 2011, 2015). We have alsoshown that mutations at S911 and T912 affected PolyPmetabolism and phosphate distribution among PolyP1-5fractions (Tomashevski & Petrov, 2015). Results describedhere extend our study towards interaction of PolyP metabol-ism and the enzyme regulation at S911 and T912 phosphor-ylation sites. Replacement by Ala led to removingphosphosite of phosphoester type and by Asp, to alteringsite to acylphosphate type. Yeast cells carrying wild type(NY13) and mutant ATPases (S911A, S911D and T912D) weregrown until mid-log phase, harvested and incubated with orwithout 2% glucose. Then PolyP were extracted and plasmamembranes were obtained to assay ATPase activity. Wild-type ATPase underwent glucose-dependent activation by 9-

fold, while mutant enzymes exhibited already elevated basalactivity by 1.6- to 10.2-fold. S911D ATPase was constitutivelyactivated by ca. 10-fold regardless the presence of glucose,whilst S911A and T912D exposed ability to be additionallyactivated by 1.7-5.0-fold upon addition of glucose. Duringglucose fermentation, orthophosphate (Pi) content dropped3 to 4 times in all strains as compared to starvation modepointing to its use for phosphorylation; however, S911Dmutant with changed type of phosphorylation had signifi-cantly elevated Pi content in both the absence and presenceof glucose (Figure 1). This points either to inability of alteredacylphosphate site at D911 to be phosphorylated or, morelikely, to irreversible phosphorylation at this site, whichshould disturb cascade of reactions ignited by glucose fer-mentation. PolyP content in the wild-type strain did not dif-fer much in the presence and absence of glucose; in mutantstrains, there was a difference seen with the most pro-nounced effect for the shortest PolyP1 fraction, whichdecreased in S911A and T912D and completely disappearedin S911D in the absence of glucose; glucose additionbrought PolyP1 amount up to control level and higher inthe case of S911D. This strain had also the most noticeablechanges in PolyP2-5 fractions, which were higher comparingto the wild type. This points to involvement of PolyP directlyor indirectly—through Pi pool—in phosphorylation.Presented data allow to suggest interaction between ATPasefunctioning and PolyP metabolism; removal of phosphositeat S911 or altering its type causes changing in Pi contentand redistribution between PolyP fractions, which may pointto preventing or altering Pi usage for phosphorylation bothof the Pma1 and other proteins and substances at differentsteps. Further study is needed to get helpful insights intofine mechanisms of functioning and regulating of thePma1 ATPase.

References

Azevedo, C., Livermore, T., & Saiardi, A. (2015). Protein polyphosphoryla-tion of lysine residues by inorganic polyphosphate. Molecular Cell,58(1), 71–82.

Lecchi, S., Nelson, C. J., Allen, K. E., Swaney, D. L., Thompson, K. L., Coon,J. J., … Slayman, C. W. (2007). Tandem phosphorylation of Ser-911and Thr-912 at the C terminus of yeast plasma membrane Hþ-ATPase

Figure 1 Effect of point mutations in C-terminal end of the Pma1 ATPase onthe distribution of PolyP fractions during glucose-dependent activation.


leads to glucose-dependent activation. Journal of Biological Chemistry,282(49), 35471–35481.

Petrov, V. V. (2011). Role of M5-M6 loop in the biogenesis and functionof the yeast Pma1Hþ-ATPase. Journal of Biomolecular Structure andDynamics, 28, 1024–1025.

Petrov, V. V. (2015). Point mutations in the extracytosolic loop betweentransmembrane segments M5 and M6 of the yeast Pma1Hþ-ATPase:Alanine-scanning mutagenesis. Journal of Biomolecular Structure andDynamics, 33(1), 70–84.

Tomashevsky, A. A., & Petrov, V. V. (2011). Point mutation in M9-M10loop of the yeast Pma1Hþ-ATPase affects both ATPase functioningand polyphosphate distribution. Journal of Biomolecular Structure andDynamics, 28, 1025–1026.

Tomashevski, A. A., & Petrov, V. V. (2013). Point mutations in the yeastPma1Hþ-ATPase affects polyphosphate (PolyP) distribution. Journal ofBiomolecular Structure and Dynamics, 31, 124–125.

Tomashevski, A. A., & Petrov, V. V. (2015). Mutations in the yeastPma1Hþ-ATPase regulatory domain affects polyphosphate metabol-ism. Journal of Biomolecular Structure and Dynamics, 33(Suppl),105–106.

108. Conformational transition ofcatalytic domain in hGMPR enzymefrom native to ligand bound state:a MD simulation study

Hridoy R. BairagyaDepartment of Biophysics, All India Institute of Medical Sciences,New Delhi 110029, India [email protected]

The human Guanosine-50-Monophosphate Reductase(hGMPR) enzyme involves in inosine biosynthesis pathway byconverting GMP to IMP and ammonia with concomitant oxi-dation of NADPH and plays a vital role in re-utilization offree intracellular bases. Type I hGMPR is expressed in all cellswhereas isoform-II can promote monocytic differentiation of

HL-60 leukemia cells. So, hGMPR-II attracts an excellentpotential target for design of isoform-specific antileukemicagent. The catalytic domain of hGMPR is a homo-tetramerwith 37-kDa subunits and monomer of this enzyme has a(a/b)8 structure of TIM barrel. The polypeptide chain of thisdomain has folded to form two separate nucleotide bindingpockets (Figure 1a). One of them accommodates cofactorNADP (di-nucleotide) and the other binds to substrate GMP(mono-nucleotide). Few crystal structures of hGMPR areobserved in different conformations. The analysis of crystalstructures explores that active site residue or Cys186 attacksC2 of substrate (GMP) to create an intermediate(Cys186SG—C2GMP) whereas ammonia leaving group is acti-vated by proton relay through Thr188 and Glu289 dyad. So,interaction between Cys186 and Thr188/Glu289 is Cys186—GMP—Glu289/Thr188 in ligand bound state (Figure 1d).Consequently, cofactor or NADPH is stabilized by Lys78,Thr105, Ser270 and Arg286/Tyr285 (Figure 1b). In spite ofthis distinction, no studies have been carried out on catalyticdomain to identify the new conformation of native state ofhGMPR enzyme. To investigate the dynamic behavior ofnative conformation of catalytic domain, molecular dynamicssimulation was carried out using CHARMM27 force field withNanoscale Molecular Dynamics (v.2.9) program In fact, MDresult suggest that 22 water molecules are observed tooccupy at position of GMP and NADPH binding region (inunliganded form). The Cys186, Thr188, Glu289 of substrateand Lys78, Glu289, Ser270, Arg286/Tyr285 of cofactor bind-ing residues move away from their respective binding pocketduring MD simulation and stabilized by conserved watermediated interactions. Four water molecules (Cys186—W1—W2—W3—W4—Thr188/Glu289) at substrate binding region(Figure 1e) and eighteen water molecules (Lys78—W5—W6—W7(—Thr105)—W8—W9—W10—W11—W12—W13—OB(Arg286)—W14—W15—W16—W17—W18—W19—OG

Figure 1. (a) GMP with NADPH bound conformation. (b, d) Residues of NADPH and IMP binding pocket in X-ray structure. (c, d) Water-mediated H-bonding inter-action of NADPH and IMP binding pocket in native conformation of MD structures.


(Ser270)—W20—W21—W22—Lys78) at cofactor bindingpocket (Figure 1c) are observed to stabilize in unligandedstate of catalytic domain, but they move and vacate thosepositions when enzyme (hGMPR)-substrate cofactor-complexis formed. Our computational study highlights structuraland functional importance of water molecules in catalyticdomain of hGMPR enzyme and suggests a rethink of con-ventional definition of chemical geometry of inhibitor bind-ing site.

Funding

This research has been supported by C.S.I.R. for SeniorResearch Associateship (Pool Scientist scheme).

109. Deficiency of thiamine shiftsmetabolism toward the a-keto acidsoversynthesis in Yarrowialipolytica yeast

Igor G. Morgunov and Svetlana V. KamzolovaG.K. Skryabin Institute of Biochemistry and Physiology ofMicroorganisms RAS, Pushchino 142290, Moscow region, Russia

[email protected]

Earlier, we found that the inhibition of isocitrate lyase byoxalic and itaconic acids shifts yeast metabolism towards thethreo-Ds-isocitric acid synthesis, a biologically active com-pound, an intermediate of Krebs cycle (Kamzolova &Morgunov, 2015; Kamzolova, Allayarov, Lunina, &Morgunov, 2016).

The purpose of this work was to study the effect of lim-ited growth of Yarrowia lipolytica yeast by a thiamine, whichis essential for multiple biochemical pathways involved inglucose catabolism.

It was shown that the growth curve of Y. lipolytica VKM Y-2378 had three phases: exponential phase (up to 12 h),retardation phase (from 12 to 24 h) caused by exhaustion ofthiamine, and stationary phase. The production of a-ketoacids (pyruvate and a-ketoglutarate) began in the growthretardation phase and continued in the stationary phase. Atthe end of cultivation, cells synthesized 41 g/L pyruvate and8g/L a-ketoglutarate. The thiamine deficiency disturbed themetabolic flow through the thiamine-dependent enzymes ofglucose catabolism. The inhibition of pyruvate dehydrogen-ase complex leads to excretion of pyruvate from the yeastcells. As the inhibition of pyruvate dehydrogenase is notcomplete, the formation of acetyl-CoA continues, providingfor the synthesis of a-ketoglutarate in Krebs cycle.a-Ketoglutarate is not oxidized in Krebs cycle since thiaminedeficiency limits a-ketoglutarate dehydrogenase, and henceit is excreted from the yeast cells. The deficiency of energyat the level of thiamine-dependent enzymes affected therespiratory chain of cells. To maintain the with induceda-keto acids production, the cells activated the initial reac-tions of glucose catabolism and Krebs cycle. It is confirmed

by increased activities of hexokinase, glucose-6-phosphatedehydrogenase, fructose bisphosphate aldolase, pyruvate kin-ase, citrate synthase and aconitate hydratase; oxaloacetate inthis case is presumably resynthesized by means of pyruvatecarboxylase reaction.

Similar effect of a thiamine deficiency has been previouslydemonstrated for Blastobotrys adeninivorans grown on glu-cose (Kamzolova & Morgunov, 2016b) and Y. lipolytica VKMY-2378 grown on glycerol-containing waste of biodiesel pro-duction (Kamzolova & Morgunov, 2018), an active superpro-ducers of a-keto acids. Therefore, it can be suggested thatthis phenomenon is a common regularity associated withthe limitation of the activity (or synthesis) of enzymesinvolved in carbohydrate utilization and a-ketoacids production.

References

Kamzolova, S. V., & Morgunov, I. G. (2015). Inhibition of isocitrate lyaseshifts metabolism toward the isocitrate overproduction in yeastYarrowia lipolytica. Journal of Biomolecular Structure and Dynamics,33(Supp1), 107–108. doi:10.1080/07391102.2015.1032802

Kamzolova, S. V., Allayarov, R. K., Lunina, J. N., & Morgunov, I. G. (2016).The effect of oxalic and itaconic acids on threo-Ds-isocitric acid pro-duction from rapeseed oil by Yarrowia lipolytica. BioresourceTechnology, 206, 128–133. doi: 10.1016/j.biortech.2016.01.092.

Kamzolova, S. V., & Morgunov, I. G. (2016). Biosynthesis of pyruvicacid from glucose by Blastobotrys adeninivorans. AppliedMicrobiology and Biotechnology, 100(17), 7689–7697. doi: 10.1007/s00253-016-7618-1.

Kamzolova, S. V., & Morgunov, I. G. (2018). Biosynthesis of pyruvic acidfrom glycerol-containing substrates and its regulation in the yeastYarrowia lipolytica. Bioresource Technology , 266, 125–133. doi:10.1016/j.biortech.2018.06.071.

110. Genome-wide survey of proteinstructural domains and analysis ofdomain architectures

Meenakshi S. Iyer, Adwait G. Joshi andR. SowdhaminiNational Centre for Biological Sciences, TIFR, GKVK, Bellary Road,Bangalore 560065, India [email protected]

In the past few years, the increase in genome and transcrip-tome studies has resulted in a plethora of protein sequencesbeing deposited in the sequence databases. But, there existsa huge deficit between the number of structures andsequences available. In this work, we have tried to bridgethis gap using SCOP superfamily domain sequences to iden-tify homologues in nonredundant database (from NCBI) (Iyer,Joshi, & Sowdhamini, 2018). The sequences were validatedusing structure-based sequence alignment HMMs derivedfrom SCOP (v1.75) superfamily members in PASS2.4 database.Domain architectures were computed for the validated hitsat structure level (SCOP) and sequence level (Pfam). Theassociated domains in a domain architecture were found tobe involved in the same biological process. A


correspondence of Pfam and SCOP superfamilies was obtainedfor each superfamily covering about 61% of Pfam families. Thestructural census (Chothia, 1992; Caetano-Anoll"es, Wang,Caetano-Anoll"es, & Mittenthal, 2009) was revisited and distri-bution of homologues across superfamilies, folds and classeswere analyzed. About 27% of NR database and 41% of the tax-onomy database (from NCBI) were covered in the study. Theresults from the above analysis have been presented in theform of a database named GenDiSþ (Pugalenthi, 2005; Iyer,2018). Profiles derived from the alignments of superfamilyhomologues can be used in sequence searches and for assign-ing structural domains to sequences (Figure 1).

Funding

This research has been supported by NCBS, TIFR.

References

Caetano-Anoll"es, G., Wang, M., Caetano-Anoll"es, D., & Mittenthal, J. E.(2009). The origin, evolution and structure of the protein world.Biochemical Journal, 417(3), 621–637.

Chothia, C. (1992). Proteins. One thousand families for the molecularbiologist. Nature, 357(6379), 543–544.

Iyer, M. S., Joshi, A. G., & Sowdhamini, R. (2018). Genome-wide survey ofremote homologues for protein domain superfamilies of known struc-ture reveals unequal distribution across structural classes. MolecularOmics, 14(4), 266–280.

Iyer, M. S. (2018). GenDiSþ: improved sequence search and validation andupdate of features for homologues of Structural Superfamily members.(Manuscript under review)

Pugalenthi, G. (2005). GenDiS: Genomic distribution of protein structuraldomain superfamilies. Nucleic Acids Research, 33, D252–D255.

111. Germination response of Triticumaestivum seeds to different exposuretimes of low-intensity EMI treatment

Gayane H. Poghosyan and Zhanna H. MukhaelyanDepartment of Biophysics, Yerevan State University, Yerevan,Armenia [email protected]

It is known that exposure to electromagnetic fields (EMF) ofmillimeter range or extremely high frequencies (EHF) hasbeneficial effects on seed germination and plant growth(Naueiene et al., 2017). This application in contrast to chem-ical methods of seeds pre-germination treatment is noninva-sive and environmentally appropriate technology what isvery important for farming industry.

The study aimed to investigate the effects of pre-sowingseed treatment with physical factor-EHF EMF (50.3 GHz fre-quency, 3–10min) on seed germination and seedling per-formance. For this, seeds of wheat (Triticum aestivum L. of‘Bezostaya’ variety) were imbibed in water for 12 hour thentreated once with EMF with 50.3 GHz frequency, for 3, 5and 10min., then left to germinate on wet filter paper inPetri dishes at 23 #C in the dark for 8 days. The irradiationwas performed using the generator G4-141 type (StateScientific-Production Enterprise ‘Istok’, Russia) with workinginterval of 37.50–53.57 GHz and power flux density 64 mWt/cm2. The germination rate, seedling length and freshweight were determined at the third and seventh day afterseeds sowing. Our findings show that the most positivegermination effects were in short-time EMI treatments (3and 5min, respectively) groups. The germination testsrevealed that EMI-treatments induced increase in germin-ation rate in seeds as compared to control. Thus, the ger-mination rate at the third day after sowing was significantly(p< 0.05) higher (by 12% and by 9%) for EMF-exposedseeds, while on the seventh day these indexes did not sig-nificantly differ from control. On the other hand, the longertime EMI –treatment (10min) did not change germinationrate for both studied days after sowing. Data also showthat seedlings grown from seeds EMI-treated (5 and 10min)groups had increased seedling weight (up to 12%) andlength (up to 8%) compared to control.

References

Naueiene, Z., & Zukiene, R. (2017). Transgenerational stress memory in E.purpurea: Seed treatment with cold plasma and electromagnetic fieldinduces changes in germination, Abstract Book, 1st Inter. Conf. “SmartBio”, Kaunas, p.159.

Figure 1.


112. Intermediate stage of thetransition of prebiotic microsystemsto life

Vladimir N. KompanichenkoInstitute for Complex Analysis RAS in Birobidzhan, 4, SholomAleyhem Str, Birobidzhan 679016, Russia [email protected]

Following the author’s inversion concept, the primary formsof life arise through thermodynamic inversion in non-livingprebiotic microsystems, when the contributions of freeenergy (F) and information (I) become prevalent over thecontribution of entropy (S) (Kompanichenko, 2017). In thisway the ‘thermodynamic corridor’ for any chemical scenarioof life origin is substantiated: Fþ I< S (prebiotic microsys-tem) ! Fþ I) S (intermediate stage, inversion moment) !Fþ I> S (primary form of life). Prebiotic organic microsys-tems can achieve the intermediate stage only under far fromequilibrium conditions, including mandatory high-frequencyoscillations of physico-chemical parameters (pressure, tem-perature and chemistry) in hydrothermal environments. Suchoscillations are considered as the fourth required conditionfor life origin, in addition to the three well-known ones:availability of organic matter, aqueous medium and a sourceof energy. A prebiotic organic microsystem at the intermedi-ate stage is characterized by internal oscillations around thepoint of bifurcation and relative efficiency of chemical reac-tions producing free energy and entropy (Figure 1). A mainidea for discussion is that self-maintained life processes werelaunched through continuous response of prebiotic microsys-tems to incessant physic-chemical oscillations in the outsideworld (stress), in connection with the Le Chatelier principle.No substantial fluctuations in a medium (for instance, inquiet ocean), no incessant recombination of (macro)mole-cules within containing organic microsystems and no per-spective for their transformation into living units. Theoscillating microsystem at the intermediate stage is unstable:further it can either be transformed into a primary livingunit through thermodynamic inversion, or degrade due to

irreversible rise of entropy. It is proposed the next step ofexperiments on the origin of life: various types of prebioticmodels should be explored under multilevel and high-fre-quency oscillations of physico-chemical parameters inthe chamber.

References

Kompanichenko, V. N. (2017). Thermodynamic Inversion: Origin of LivingSystems. Cham (Switzerland): Springer International Publishing.https://link.springer.com/book/10.1007/978-3-319-53512-8.

113. MD simulation based study tounderstand the role of pre-hydrolysisnetwork in the comparative dynamicsof A59G-H-Ras and wild-type H-Ras

Neeru Sharma, Uddhavesh B. Sonavane andRajendra JoshiHPC-M&BA Group, Centre for Development of AdvancedComputing, Panchawati Road, Pune, 411008, India

[email protected]

The Ras family of proteins plays an important role in cell-pro-liferation, differentiation, apoptosis, membrane localizationand ultimately signal transduction (Downward, 2003). Theprotein Ras has three isoforms namely H-Ras, K-Ras and N-Ras and belongs to a class of G-proteins. In normal condi-tions, wild-type Ras regularly cycles between active GTPbound and inactive GDP bound conformations. Also being aGTPase, Ras also possess an intrinsic capability to hydrolyzeGTP to GDP and maintain a highly controlled regulationbetween active and inactive states (Lu et al., 2016). In gain-of-function mutations of Ras, the intrinsic hydrolysis activityas well GAP-assisted GTP hydrolysis activity is hindered andhence the protein tends to stuck in the permanently activeoncogenic state. The oncoprotein Ras is found to be mutatedin more than 30% of human cancers (Adjei, 2001). The

Figure 1. Intermediate state between ‘non-life and life’.


present work aims to explore the dynamics of the wild-typeand mutant A59G-HRas using long-time scale MD simula-tions, followed by advance MD analytics techniques likeMSM based analysis. The in-depth insight into the crucialinteractions of wild-type and mutant, especially for the pre-hydrolysis network is reported in the study. Conventional aswell feature-based PCA analyses have also been performedto differentiate the wild-type and mutant conformations. Thestudy focuses on the relation between disruption of the cru-cial pre-hydrolysis network and the oncogenic conformationin the mutant A59G-HRas ensemble. Pivotal roles of residuesfrom SwI, SwII and P-loop have been reported.

References

Adjei, A. A. (2001). Blocking oncogenic Ras signaling for cancer therapy.Journal of the National Cancer Institute, 93(14), 1062–1074.

Downward, J. (2003). Targeting RAS signalling pathways in cancer ther-apy. Nature Reviews Cancer, 3(1), 11.

Lu, S., Jang, H., Muratcioglu, S., Gursoy, A., Keskin, O., Nussinov, R., &Zhang, J. (2016). Ras conformational ensembles, allostery, and signal-ing. Chemical Reviews, 116(11), 6607–6665.

114. Molecular modelling anddocking studies of glomalin relatedsoil protein from Rhizophagusirregularis

Dipti Mothay and K. V. RameshDepartment of Biotechnology, Jain University, School of Sciences,#18/3, 9th Main Road, Jayanagar East, Jaya Nagar 3rd Block,Bengaluru 560041, Karnataka, India

[email protected]

Glomalin-related soil protein (GRSP), is an insoluble glyco-protein, produced in copious amounts on hyphae of arbus-cular mycorrhizal fungi (AMF). It provides unique propertiesto the soil such as mechanical stability, amelioration of salin-ity and aggregate water stability. Due to high sequence simi-larity of GRSP sequence identified from Rhizophagusirregularis, a mycorrhizal fungus, with heat shock proteins 60(Hsp60) of various other species, this protein is said to be aputative homolog of Hsp60. There is a lack of evidence atboth the experimental and theoretical level with respect tothe structural information of GRSP and its interaction withhumic acid, one of the components of soil organic matter.Thus, the present study has focussed on evolutionary studiesof Hsp60, followed by homology based 3D modeling andmolecular docking of GRSP with humic acid.

Based on the phylogenetic studies, it is evident that Hsp60 pro-tein from R. irregularis from Glomeromycota shares a very closeevolutionary relationship with fungi belonging to Ascomycota andBasidiomycota, with classical a-Proteobacteria as the commonancestor. Comprehensive analysis of mitochondrial hsp60 fromselected fungal taxa from the evolutionary point of view explainsthe possibility of gene duplication and or horizontal gene transferof this gene across various fungal species. Molecular clock analysiscarried out by BEAST programme for the 14 common protein cod-ing genes (inclusive of hsp60), identified from 21 mitochondrialgenomes belonging to fungal divisions Ascomycota,Basidiomycota and Glomeromycota estimates their time for diver-sification to be around 1425 Mya. Population genetics study cred-ibly explains high genetic variability associated with fungal hsp60presumably brought by random genetic recombination events.The results also confirm a high level of genetic differentiation ofhsp60 among all the three fungal populations analyzed.

PsiBlast search and Swiss-Model server identified Hsp60 fromChlorobaculum tepidum (5DA8_A) as the potential template forgenerating 3D model of GRSP. Accuracy of the 3D model wassubsequently validated using Ramachandran plot analysis, ERRAT,ProCheck and Verify 3D. Among the two identified N-Linked gly-cosylation sites, the one that was present towards the C-terminalend was included in the truncated 94 residue fragment of the 3Dmodel. Using Glycam Server, a glycoprotein model of GRSP wasbuilt by attaching the carbohydrate moiety rich in mannose, gal-actose and N-acetyl galactosamine to the asp94 residue of thetruncated model. AutoDock software was successful in dockingthe standard 3D model of leonardite humic acid (chain L and U),onto the GRSP- carbohydrate complex. This was followed bydocking the ‘GRSP glycoprotein—humic acid’ complex with 50water molecules. These findings open up new avenues for explor-ing the interactions of glomalin related soil protein with the soilorganic matter, and its influence in aggregating the water mole-cules in improving soil productivity.

References

Gadkar, V., & Rillig, M. C. (2006). The arbuscular mycorrhizal fungal pro-tein glomalin is a putative homolog of heat shock protein 60. FEMSMicrobiology Letters, 263(1), 93–101.

Petrov, D., Tunega, D., Gerzabek, M. H., & Oostenbrink, C. (2017).Molecular dynamics simulations of the standard leonardite humicacid: Microscopic analysis of the structure and dynamics.Environmental Science & Technology, 51(10), 5414–5424.


115. Sex-dependent differences inpoly(ADP-ribose)polymerase 1 activityof rat liver and thymocyte nuclei

Karine S. Matinyan, Anush L. Asatryan, Irina G.Artsruni, Arshak A. Karapetyan andEmil S. GevorgyanDepartment of Biophysics, Faculty of Biology, Yerevan StateUniversity, Yerevan 0025, Armenia [email protected]

Sex-dependent differences among therapeutic effects ofPARP inhibitors were documented in treatment of variouspathologies. The purpose of this study was to examinewhether PARP 1 activity displays sexual dimorphism in livercells and thymocytes of rat (Virag & Szabo, 2002; Waxman &Holloway, 2009). PARP 1 inhibition by benzamide (Bam) andATP in liver and thymocyte nuclei after in vivo administrationof cis-diammine-1,1-cyclobutanedicarboxylate platinum (cis-DDP) to animals was studied. Our data show that femalethymocyte and liver nuclei, in contrast to male counterparts,exhibit organ-specific differences in PARP 1 activities. PARP 1activity of female thymocyte nuclei was unaffected by Bam,while in liver nuclei it was suppressed by 45%. PARP 1 activ-ity of the male rats liver nuclei was completely suppressedby Bam. It was demonstrated that in liver nuclei of male ratstreated with cis-DDP PARP 1 activity decreased more than 3times, whereas in liver nuclei of females it was suppressedonly by 45%. In addition, the studies revealed that thymo-cyte nuclei of both sexes were more resistant to in vivo andex vivo chemical interventions than liver nuclei. It was shownthat in vivo administration of anti-cancer drug cis-DDP iscapable to modulate PARP 1 inhibition by Bam in liver nucleiin sex-dependent manner which should be considered forimproving therapeutic outcomes in adjuvant cancer chemo-therapy by PARP 1 inhibitors.

References

Virag, L., & Szabo, C. (2002). The Therapeutic Potential of Poly(ADP-Ribose)Polymerase Inhibitors. Pharmacological Reviews, 54(3), 375–418.

Waxman, D. J., & Holloway, M. G. (2009). Sex Differences in theExpression of Hepatic Drug Metabolizing Enzymes. MolecularPharmacology, 76(2), 215–228.

116. The distribution of electrostaticsurface potentials is an importantfactor underlying the temperatureadaptation of subtilisin-likeserine proteases

Yuan-Ling Xia, Xiao-Yan Zhang, Yan Tao andShu-Qun Liu

State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan & School of Life Sciences, Yunnan University,Kunming 650091, China [email protected]

The molecular mechanisms of enzymatic temperature adap-tation are dictated by the delicate balance between the sta-bility, flexibility and activity of the extremophilic enzymes;therefore, identifying the factors that rule the stability–flexi-bility–activity relationship is essential for both fundamentalresearch and industrial applications (Papaleo, Tiberti,Invernizzi, Pasi, & Ranzani, 2011). To investigate the role ofelectrostatics in temperature adaptation, we have performeda comparative study on three differently temperature-adapted subtilisin-like serine proteases, the psychrophilicVPR, the mesophilic PRK and the thermophilic AQN, usingmultiple-replica molecular dynamics simulations followed bycontinuum electrostatics calculations and comparison of elec-trostatic surface potentials. Comparison of the salt-bridgeelectrostatic strength at 300 K reveals that they on averageprovide greater stabilization to VPR and PRK than to AQN,indicating that salt bridge may not be a crucial factor indetermining the overall thermostability of subtilisin-like ser-ine proteases. The most significant difference lies in the dis-tribution of different electrostatic surface potentials,especially across the back surfaces, that is, those of PRK andAQN are dominated by the electro-positive potential withsporadic electro-neutral patches interspersed, and VPR’s backsurface is dominated by the electro-negative potential,although the catalytic centers (on the front surface) of thethree proteases collectively feature the electro-negativepotential. Because water is a protic solvent, it interacts morefavorably with the electro-negative surface than with theelectro-positive and neutral surfaces, and this explains why aprotein with more negative surface charge is more soluble inwater than its homologue with more positive surface charge.Furthermore, since water molecules around the negativelycharged and polar uncharged surfaces form a high-density,collapsed structure with weak hydrogen-bonding interactionsand fast dynamics, they would facilitate conformational fluc-tuations, hence enhancing the local flexibility of the electro-negative surface regions. On the contrary, the low-density,expanded structure of hydration shell formed around thepositively charged and nonpolar/neutral surfaces wouldrestrain their fluctuations and hence increase the local rigid-ity. For the mesophilic PRK at 300 K, comparison of the Caroot-mean-square-fluctuation values averaged over the solv-ent-exposed residues covered by the electro-negative, elec-tro-positive and electro-neutral potentials reveals that theelectro-negative surface is indeed more flexible than theelectro-positive and nonpolar/neutral surfaces. For all threeproteases, the electro-negative surface potential around theactive center could provide the active-center flexibility neces-sary for nucleophilic attack and proton transfer. For VPR, thepredominant distribution of the electro-negative potential onthe back surface could ensure sufficient low-temperaturesolubility and surface flexibility crucial for VPR’s low-tempera-ture catalytic activity. For AQN, the predominant distributionof the electro-positive potential likely contributes to


enhancing the surface rigidity necessary for the mainten-ance of the structural integrity at high temperatures.Therefore, differently charged surface patches can affect/modulate via differential interactions with water moleculesthe protein solubility and the local structural flexibility/rigidity (Xia et al., 2018) and, therefore, the electrostaticsurface potential distribution is an important factor under-lying the temperature adaptation of subtilisin-like ser-ine proteases.

Funding

This research has been supported by NSFC of China (nos.31160181 and 31370715) and Programs for Excellent YoungTalents and Donglu Scholar in the Yunnan University.

References

Papaleo, E., Tiberti, M., Invernizzi, G., Pasi, M., & Ranzani, V. (2011).Molecular determinants of enzyme cold adaptation: Comparativestructural and computational studies of cold- and warm-adaptedenzymes. Current Protein & Peptide Science, 12, 657–683.

Xia, Y.-L., Sun, J.-H., Ai, S.-M., Li, Y., Du, X., Sang, P., … Liu, S.-Q.(2018). Insights into the role of electrostatics in temperature adapta-tion: A comparative study of psychrophilic, mesophilic, and thermo-philic subtilisin-like serine proteases. RSC Advances, 8(52),29698–29713.

117. The mutual orientation betweenV1/V2 and V3 is the structuraldeterminant of HIV-1 gp120conformational states and dynamics

Yi Li, Lei Deng, Yan Tao and Shu-Qun LiuState Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan & School of Life Sciences, Yunnan University,Kunming, 650091, China [email protected]

The trimeric envelope spike (Env), which is the infectionmachinery of HIV-1 and the sole target of protective neutral-izing antibodies, is composed of three external glycoproteingp120 subunits associated non-covalently with three trans-membrane glycoprotein gp41 subunits. The entry of HIV-1 isinitiated by binding of gp120 to the cell-surface receptorCD4, which induces substantial changes in gp120 conform-ation involving the reorientation of the V1/V2 region relativeto the V3 loop, rearrangements of the bridging-sheet ele-ments, and formation and exposure of the coreceptor-bind-ing site, ultimately leading to the transition of Env from anunliganded ‘closed’ state to a CD4-bound ‘open’ conform-ation (Guttman et al., 2014). Although structural studies haveprovided valuable information on various states of Env/gp120, the detailed questions on many dynamic aspects ofgp120, such as what the differences in conformationaldynamics and thermodynamics are between the unliganded

and CD4-bound states, which structural elements determinethese differences, and how gp120 regulates transitionbetween different states, still remain answered. To answerthese questions, we have performed a series of multiple-rep-lica molecular dynamics simulations on the three structuralmodels (i.e., the unliganded gp120, the CD4-free gp120 inthe CD4-bound state and the gp120-CD4 complex), with thecumulative simulation time length reaching 3 ms.Comparative analyses of their respective concatenated equi-librium trajectories in terms of structural deviation and con-formational flexibility reveal that the CD4-free and CD4-complexed gp120s are more structurally mobile and confor-mationally flexible than the unliganded gp120. The mostpronounced differences in the local flexibility between theunliganded and CD4-complexed/free gp120s were observedin the V1/V2 region and V3 loop, which exhibit significantlyhigher Ca root-mean-square-fluctuation values in the latterthan in the former due to the dissociation between and fullexposure of V1/V2 and V3. Analyses of the largest-amplitudemotion modes indicate that in the CD4-free gp120, themutual approach between V1/V2 and V3 could lead to theconformational transition to the unliganded state.Comparison of the constructed free-energy landscapes(FELs) shows that CD4-complexed and CD4-free gp120shave larger conformational entropy, richer conformationaldiversity and lower thermal stability than the unligandedgp120. Further comparison of the representative structuresextracted from the free energy basins of FELs reveals thatthe presence of CD4 weakens the reorientation ability ofV1/V2 relative to V3 and therefore restricts the conform-ational transition from the CD4-bound state to the unli-ganded state. Therefore, it can be concluded that themutual orientation of the V1/V2 region with respective tothe V3 loop is not only a major marker for distinguishingbetween the unliganded and CD4-bound states, but deter-mines also the differences in the conformational dynamicsand thermodynamics between these two states. Lockinggp120 conformation via restraining mutual reorientationsbetween V1/V2 and V3 with small molecules seems to bea promising strategy to control HIV-1 infection (Li, Deng,Yang, Sang, & Liu, 2019).

Funding

This research has been supported by NSFC of China (nos.31370715 and 31160181) and Programs for Excellent YoungTalents and Donglu Scholar in the Yunnan University.

References

Guttman, M., Garcia, N. K., Cupo, A., Matsui, T., Julien, J.-P., Sanders,R. W., … Lee, K. K. (2014). CD4-Induced activation in a solubleHIV-1 Env trimer. Structure, 22(7), 974–984.

Li, Y., Deng, L., Yang, L. Q., Sang, P., & Liu, S. Q. (2019). Effects of CD4binding on conformational dynamics, molecular motions, and thermo-dynamics of HIV-1 gp120. International Journal of Molecular Sciences,20(2), 260.


118. Understanding the basis forprotective role of E219K prion variantusing molecular dynamics simulations

Vinod Jani, Uddhavesh Sonavane and Rajendra JoshiHigh Performance Computing-Medical & BioinformaticsApplication Group, Centre for Development of AdvancedComputing (C-DAC), C-DAC Innovation Park, Panchavati, Pashan,Pune 411007, India [email protected]@cdac.in

The scrapie form of prion protein is responsible for variousneurodegenerative diseases. Mutation in prion protein is oneof the factors which leads to pathogenic conversion of normalprion PrPc protein to scrapie form PrPSc (Friedman-Levi et al.,2011). But, previous studies on the prion protein also suggestthat E219K prion mutant (glutamate to Lysine mutation at resi-due position 219) is more stable than the wild type protein(Biljan et al., 2012; Jahandideh, Jamalan, & Faridounnia, 2015).Hence, in this study the wild type prion protein, prion mutantE200K (glutamate to Lysine mutation at residue position 200)and E219K, a naturally occurring mutant, which is consideredto protect against Creutzfeldt-Jakob disease (CJD) have beenstudied. In order to understand the structural changes, wehave carried out detailed atomistic simulation of three sys-tems. Principal component analysis, free-energy analysis,dynamic cross correlation analysis and other analyses havebeen carried out. Our analysis shows that the extra stability ofE219K mutant is result of increase in number of native con-tacts, strong hydrogen bond network and less randommotions. The pathogenicity of E200K may be the result of lossof some crucial salt–bridge interaction and anti-correlatedmotion between helix 2(H2) and helix 3 (H3) and increase insolvent accessible surface area of hydrophobic residues. Thus,this study shows that native contacts, salt–bridge interactionand hydrogen bonds between H2 and H3 play an importantrole in the maintaining stability/pathogenicity of the prion pro-tein (Figure 1).

References

Biljan, I., Giachin, G., Ilc, G., Zhukov, I., Plavec, J., & Legname, G. (2012).Structural basis for the protective effect of the human prion protein

carrying the dominant-negative E219K polymorphism. BiochemicalJournal, 446(2), 243–251.

Friedman-Levi, Y., Meiner, Z., Canello, T., Frid, K., Kovacs, G. G., Budka, H.,… Gabizon, R. (2011). Fatal prion disease in a mouse model of gen-etic E200K Creutzfeldt-Jakob disease. PLoS Pathogens, 7(11), e1002350.

Jahandideh, S., Jamalan, M., & Faridounnia, M. (2015). Molecular dynam-ics study of the dominant-negative E219K polymorphism in humanprion protein. Journal of Biomolecular Structure and Dynamics, 33(6),1315–1325.

119. Thermodynamic stability andmolecular dimensions of pseudoknots

Calliste Reiling-Steffensmeier and Luis A. MarkyDepartment of Pharmaceutical Sciences, College of Pharmacy,University of Nebraska Medical Center, Omaha, NE, USA

[email protected]

Pseudoknots are prevalent RNA structures that play signifi-cant roles in the biological function of RNA. In this work, wehave investigated the unfolding thermodynamics and solu-tion molecular dimensions of two pseudoknots with stemloop complementarity to model the frameshift occurrence ofribosomal pseudoknots.

We used a combination of UV and DSC melting techni-ques and SAXS to determine the thermodynamic stabilityand solution molecular dimensions of two pseudoknotswith sequences: d(TCTCTT1111AAAAAAAAGAGAT5TTTTTTT)(PsK11-DNA) and r(UCUCU-U1111AAAAAAAAGAGAU5

UUUUUUU) (PsK11-RNA), ‘T11’ and ‘U1111’ are the loopsequences complementary to one stem. We used three buf-fer conditions: 10mM Sodium Phosphate (NaPi), 10mMSodium Cacodylate (NaCac) and 10mM Sodium Cacodylatewith 10mM MgCl2 (NaCac & Mg2þ).

Both pseudoknots form intramolecularly with PsK11-DNAin the ‘B’ conformation and PsK11-RNA in the ‘A’ conform-ation, determined by their circular dichroism spectra. Thesepseudoknots were slightly more stable in the NaPi buffer by0.9 kcal/mol. However, the addition of Mg2þ to the NaCacbuffer significantly increased their stability, by 10.8 (PsK11-DNA) and 8.7 kcal/mol (PsK11-RNA). Overall, PsK11-RNA ismore stable by 5 kcal/mol, due to its higher TM and unfold-ing enthalpy. However, this is reduced to 3 kcal/mol in the

Figure 1.


presence of Mg2þ. The TM-dependencies on salt yielded simi-lar ion uptakes of " 3mol Naþ/mol in both buffers and" 0.1mol of Naþ/mol in NaCac & Mg2þ. The TM-dependencieson osmolyte (ethylene glycol) yielded lower water uptakesfor PsK11-DNA by 13mol H2O/mol, in NaPi and NaCac.However, in the presence of Mg2þ PsK11-DNA has a greaterwater uptake by 10mol H20/mol.

The SAXS data indicate PsK11-DNA has a length of " 80Åand width of " 32Å, while PsK11-RNA has a length of " 65Åand width of " 30Å; both molecules have a thickness ofabout 14 Å. Thus, PsK11-RNA is more compact.

The main conclusion is that the higher compactness ofthe RNA pseudoknot is consistent with its greater thermo-dynamic stability.

Funding

This research has been supported by grant MCB-1122029from the National Science Foundation.

120. Unfolding thermodynamicsof DNA kissing-loop interactions

Carolyn E. Carr and Luis A. MarkyDepartment of Pharmaceutical Sciences, University of NebraskaMedical Center, Omaha, NE 68198-6025, USA

[email protected]

Kissing-loop interactions (KLI) are common biological motifsfound in RNA–RNA interactions. While significant computa-tional work has been done on the thermodynamics andstructure of RNA kissing-loop interactions, there are only afew experimental studies on the thermodynamics of kissingand none give complete and detailed thermodynamicreports. As a proof of concept, we modeled a monomolecu-lar and bimolecular KLI and determined a complete thermo-dynamic description of the folding/unfolding of anintramolecular DNA kissing complex (Kissing Complex), twobimolecular kissing complexes (KHp1þ KHp2, KS1þ KS2), anon-kissing control complex (No Kiss Com) and their appro-priate control stem-loop motifs (KHp1, KHp2 and TLKHp2). Wealso tested the ability of the kissing complex to engage inkissing when sterically strained by adding a complementarystrand to the base. The transition temperature of each oligo-nucleotide remains constant over a ten-fold range of totalstrand concentration, indicating all molecules formed intra-molecularly, with the exception of KHp2, which displayedtwo transitions, one bimolecular and one monomolecularwhich was attributed to the formation of a duplex. KissingComplex unfolded triphasically, with the last two peaks corre-sponding to the two hairpins that make up the kissing com-plex and the first peak corresponding to the KLI (DHcal ¼23.7 kcal/mol). No Kiss Com unfolded biphasically, with twopeaks corresponding to the two individual hairpins thatmake up the complex, but no peak relating to a KLI,

indicating that we are indeed able to detect the thermody-namics of a KLI. The two bimolecular kissing complexes alsodisplayed KLI but only at high concentrations. Addition ofthe complementary strand to the base of Kissing Complexabolished the KLI while maintaining integrity of the individ-ual hairpin stems.

Funding

This research has been supported by Grant MCB-1122029from NSF.

121. Accounting for electrostaticpolarization in gas-phase simulationsof ion mobility spectrometry

Christopher A. Myersa and Alan A. Chenb,caPhysics Department; bChemistry Department; cRNA Institute,University at Albany (SUNY), Albany, 12222, NY

[email protected]

Molecular dynamics (MD) simulations coupled with ionmobility spectrometry (IMS), a gas-phase extension to massspectrometry that further filters the analytes by their top-ology, allows researchers to perform three-dimensional struc-tural elucidation of nucleic acids. Recently, combined IMS-MD studies of short DNA duplexes (Porrini et al., 2017) andhairpins (Lippens, Ranganathan, D’Esposito, & Fabris, 2016),however, have struggled with obtaining accurately compactstructures that agree with the experimentally determinedcollision cross sections for parts of their work. Because IMSexperiments are performed on charged molecules, long-range electrostatic interactions, as well as the changes incharge density due to protonated phosphate sites along thebackbone, play a crucial role in understanding how nucleicacids behave in a gaseous environment and can benefit froma detailed quantum chemical analysis.

In the work presented here, we explore what a properlytuned electrostatic force field for nucleic acids might looklike. Based off of density functional theory calculations ofsmall oligonucleotides, we examine how one could adjustthe partial charges in an MD force field to more accuratelyreplicate Coulomb interactions for charged and protonatednucleic acids. Since the electrostatic interactions used inmost force fields are often based off of single monomerquantum chemistry data and do not account for hydrogenbonding energies, the work presented here proposes newmethods for parameterizing force fields using multiple oligo-nucleotide geometries and, more generally, how polarizationis accounted for throughout an MD simulation.

Funding

Alan A. Chen is supported by NSF award #MCB1651877.


References

Lippens, J. L., Ranganathan, S. V., D’Esposito, R. J., & Fabris, D. (2016).Modular calibrant sets for the structural analysis of nucleic acids byion mobility spectrometry mass spectrometry. The Analyst, 141(13),4084–4099.

Porrini, M., Rosu, F., Rabin, C., Darr"e, L., G"omez, H., Orozco, M., &Gabelica, V. (2017). Compaction of duplex nucleic acids upon nativeelectrospray mass spectrometry. ACS Central Science, 3(5), 454–461.

Rueda, M., Kalko, S. G., Luque, F. J., & Orozco, M. (2003). The structureand dynamics of DNA in the gas phase. Journal of the AmericanChemical Society, 125 (26), 8007–8014.

122. Binding peculiarities of ethidiumbromide to polyguanine acid

Marine A. Parsadanyana, Ara P. Antonyana, Mikayel V.Minasyantsb and Armen T. KarapetiancaDepartment of Biophysics, Yerevan State University, Yerevan,Armenia; bUWC Dilijan College, Dilijan, Armenia; cDepartment ofPhysics and Electrotechnics, Natioanl University of Architectureand Construction of Armenia, Yerevan, Armenia

[email protected]

Ethidium bromide (EtBr) binding to poly(G)4 has beenstudied using spectroscopic methods at the solution ionicstrengths 0.11, 0.31 and 0.51M. Spectral characteristics(changes of absorption and fluorescence spectra) of EtBr-poly(G)4 complexes are analogous to those obtained for EtBrcomplexes with DNA or poly(rA)-poly(rU) (Bochman,Paeschke, & Zakian, 2012). The studies were carried out atthe temperatures 293, 298 and 300 K to determine the bind-ing thermodynamic parameters. Based on the absorptionand fluorescence spectra, the binding curves in Scatchard’scoordinates were constructed at the mentioned ionicstrengths of the solution and temperatures (EtBr bindingcurves to poly(G)4 in Scatchard’s coordinates are similar tothose obtained for this ligand complexes with DNA), and thevalues of binding constant K and binding site size n, DG, DHand DS were determined by standard formulas.

Value of the enthalpy change (DH ) % 2.2 ccal/mol) isabout three-fold less, than in the case of intercalation bind-ing of EtBr to double-stranded NA and the binding constantvalue K is about an order less, than the analogous value cor-responding to EtBr intercalation into the plane of ds-NA basepairs. Most probably, it is the fact that at the mentioned con-ditions EtBr binds to poly(G)4 by semi-intercalation mode. Itis indicated by the fact that the values of DG and DS are incorrespondence to those obtained for EtBr complexes withsingle- or double-stranded DNA at the binding semi-intercal-ation mode.

The obtained data indicate that EtBr binding modes tonucleic acids depend on structural state of the latters.Particularly, in the case of NA structure dense packaging,EtBr intercalation becomes difficult and the main bindingmode remains semi-intercalation, as it was revealed for thisligand complexes with poly(G)4. Consequently, by investigat-ing interactions of synthetic four-stranded polyguanylic acidwith EtBr, we can understand the structural differences ofstudied synthetic and right-handed B- or A-conformationaltypes of nucleic acids, at the same external conditions.

References

Bochman, M. L., Paeschke, K., & Zakian, V. A. (2012). DNA secondarystructures: Stability and function of G-quadruplex structures. NatureReviews Genetics, 13(11), 770–780.

123. Clustering of stacked nucleobasesin a stretched DNA conformationenhances facilitated diffusion of DNAbinding proteins as revealed bymolecular dynamics simulations

Anupam Mondal and Arnab BhattacherjeeSchool of Computational and Integrative Sciences, JawaharlalNehru University, New Delhi, 110067, India

[email protected]

Figure 1.


DNA damage and repair refers to the creation and correctionof DNA lesions that threat genome integrity. Homologousrecombination is the common mechanism to prevent theDNA from the deleterious effects caused by mutations dueto the error in replication or induced by environmentalchanges. Experiments (Chen, Yang, & Pavletich, 2008; Chenet al., 2017) show that during the process, RecA proteinbinds with genomic DNA, which is unusually elongated(approximately 1.5 times to its contour length in B-DNA) andits nucleobases are clustered together in a specific manner.How these structural changes in DNA affect its interactionwith the protein? What are the implications of such structuraldiversity in identifying damaged DNA sites? Aided by exten-sive Langevin dynamics simulations we probe these issues,where we study the diffusion of a protein on a force-inducedstretched DNA. Upon pulling systematically at the 30 terminiof a 100 bp DNA by constant forces, the DNA exhibits a con-formational transition from B-DNA to ladder-like S-DNA con-formation via

P-DNA (Figure 1) intermediate. Our analysis

suggests that the resulting stretched DNA features non-uni-form clusters of nucleotide bases such as doublets, triplets,quadruplets and so on separated by large rise gap. We foundthat relative population of these clusters govern the rugged-ness of the protein–DNA binding energy landscape andthereby the ability of a protein to locate its target DNA sites.By analyzing different force regimes, we delineate the under-lying translocation mechanism of a DNA binding protein andunderscore the significance of triplex formation in regulatingthe protein diffusion on DNA. In my presentation, I shalldescribe our recent findings and discuss them in the light ofrecent experimental observations.

References

Chen, Z., Yang, H., & Pavletich, N. P. (2008). Mechanism of homologousrecombination from the RecA-ssDNA/dsDNA structures. Nature,453(7194), 489–494.

Chen, J., Tang, Q., Guo, S., Lu, C., Le, S., & Yan, J. (2017). Parallel triplexstructure formed between stretched single-stranded DNA and hom-ologous duplex DNA. Nucleic Acids Research, 45(17), 10032–10041.

124. Comparative physico chemicaland biological studies ofphosphorylguanidine oligonucleotidediasteriomers

Alexander A. Lomzov, Valeria S. Apukhtina,Andrey V. Shernyukov, Maxim S. Kupryushkin,Georgy Yu. Shevelev and Dmitrii V. PyshnyiInstitute of Chemical Biology and Fundamental Medicine SB RAS,Novosibirsk State University, Novosibirsk, 630090, Russia

[email protected]

Synthetic oligonucleotide analogs are widely used in variousfields of molecular biology, nanobiotechnology and medicine.Recently, we developed a new type of phosphate-modifiednucleic acids: phosphorylguanidine (PG) oligonucleotides

(PGOs). Guanidine residue can be introduced into desired posi-tions of an oligonucleotide by the standard automatic phos-phitamide protocol of DNA synthesis (Stetsenko, Kupryushkin,& Pyshnyi, 2014; Kuprushkin, Pyshnyi, & Stetsenko, 2014).

In the present work, studied the absolute configuration ofphosphate atom in PGO. First, we analyzed monomodifiedoligomer d(TpCp!A) (! - shows N,N,N0,N0-tetramethylguani-dine moiety). Reverse-phase HPLC analysis allowed to isolatetwo individual diastereomers of monomodified oligomer(fast and slow). Both diastereomers of d(TpCp!A) were notdigested with a snake venom phosphodiesterase after 7 days,whereas native analog was fully degraded after 30min.Ultraviolet and circular dichroism spectroscopy methods, aswell as 1D and 2D NMR spectroscopy showed high similar-ity of the diastereomers. Only detailed computational ana-lysis of restraint penalty energies via restrained moleculardynamics simulations allowed us to conclude that mostlikely, the fast isomer is the Sp- and the slow one is the Rp-diastereomer.

To study PGO properties more deeply we designed DNAduplex (d(50-CAGCGGCG!TG-30)/d(CACGCCGCTG)) with singleN,N0-dimethyl-N,N0-ethylguanidine modification. Using RP-HPLC we isolated individual diastereomers of PGO. 1D (1H,32P) and 2D (1H-1H NOESY, 1H-1H COSY, 1H-1H TOCSY, 1H-31PCOSY, 1H-13C HSQC, 1H-13C HMBC) NMR spectra wererecorded. After assignment of all non-exchangeable protonsin NOESY spectra, using the restrained molecular dynamicsmethod, we determined that the fast diastereomer is Rp andthe slow-Sp. The structural analysis showed that the intro-duction of the PG group into the oligomer does not disturbthe B-form of the double DNA helix.

Computer analysis of the Taq DNA polymerase complexeswith PGO duplexes and experimental 10-mer PGO primerelongation were carried out. The significant difference in theprocessing of the Rp and Sp isomers was observed. Thedetailed comparative analysis of the data obtained allowedus to determine the origin of the different processing of thediastereomers by the enzyme.

The data obtained show that the phosphorylguanidine oli-gonucleotides are promising for a number of applicationssuch as PCR analysis, biosensors (Dmitrienko et al., 2016) andtherapeutics.

Funding

This research has been supported by the Russian ScienceFoundation [grant No. 18-14-00357].

References

Dmitrienko, E., Naumova, O., Fomin, B., Kupryushkin, M., Volkova, A.,Amirkhanov, N., … Pyshnyi, D. (2016). Surface modification of SOI-FET sensors for label-free and specific detection of short RNA analyte.Nanomedicine, 11(16), 2073–2082.

Kupryushkin, M. S., Pyshnyi, D. V., & Stetsenko, D. A. (2014). Phosphorylguanidines: A new type of nucleic Acid analogues. Acta Naturae, 6 (4),116–118.

Stetsenko, D. A., Kupryushkin, M. S., & Pyshnyi, D. V. 2014. Internationalpatent application WO2016/028187 A1, August 22.


125. Comparative study of bindingof some intercalating compoundswith DNA

Poghos O. Vardevanyana, Ara P. Antonyana,Marine A. Parsadanyana, Mariam A. Shahinyana andVictoria G. SahakyanbaDepartment of Biophysics, Yerevan State University, Yerevan,Armenia; bDepartment of Clinical Pathology, Yerevan StateMedical University after M.Heratsi, Yerevan, Armenia

[email protected]

The comparative study of DNA-acridine orange (AO), DNA-ethidium bromide (EtBr) and DNA-methylene blue (MB) com-plexes has been carried out using UV melting method at thesolution ionic strength 0.02M, in the interval of ligand/DNAconcentration ratio change—0<r * 0.33. Changes of themelting parameter values (d(1/Tm) and d(DT/Tm

2)) of thesecomplexes were obtained. The obtained data indicate thatMB, AO and EtBr, being intercalators, stabilize DNA ds-struc-ture. In the case of AO this effect is conditioned both byintercalator and by electrostatic binding modes (Sarkar, Das,Basak, & Chattopadhyay, 2008), in the case of MB this effectis conditioned by semi-intercalation and electrostatic modes.Meanwhile, the stabilizing effect of MB on DNA ds-structureis higher than that of AO. This fact indicates that in the caseof AO the contribution of the electrostatic mode to ds-DNAstabilization is much weaker, than in the case of MB. In thecase of EtBr, a weakening of DNA ds-structure stabilization isfound out, which is conditioned by the fact that EtBr showsa semi-intercalation type of the binding to ss-structures ofDNA as well, which becomes significant and invokes a desta-bilization of its native structure.

The interaction of EtBr, MB and AO with DNA at the solu-tion ionic strength 0.02M has been studied by differentialspectroscopy method as well. The results of these studiesalso indicate that EtBr binds to DNA by intercalation, semi-intercalation and electrostatic modes; MB—by semi-intercal-ation and electrostatic modes and AO—by intercalation andelectrostatic modes, as in case of interaction of these ligandswith poly(rA)-poly(rU) (Vardevanyan, Antonyan, Parsadanyan,Shahinyan, & Sahakyan, 2017). It was shown that the simul-taneous performance of intercalation and semi-intercalationmodes conditions the formation of a real isosbestic point (IP)in the absorption spectra of EtBr-DNA complexes. In theabsence of intercalation at MB interaction with DNA apseudo-IP in differential absorption spectra of these com-plexes is formed. In the spectra of AO-DNA complexes bothIP and pseudo-IP are not formed, despite the fact that thisligand binds to DNA by intercalation mode.

References

Sarkar, D., Das, P., Basak, S., & Chattopadhyay, N. (2008). Binding inter-action of cationic phenazinium dyes with calf thymus DNA: A com-parative study. The Journal of Physical Chemistry B, 112(30),9243–9249.

Vardevanyan, P. O., Antonyan, A. P., Parsadanyan, M. A., Shahinyan,M. A., & Sahakyan, V. G. (2017). Peculiarities of interaction of syntheticpolyribonucleotide poly(rA)-poly(rU) with some intercalators. Journalof Biomolecular Structure and Dynamics, 35, 1–7. doi: 10.1080/07391102.2017.1402708.

126. Concentrational dependenceof melting temperature: possibleexplanation of non-monotonic behavior

A. Asatryan, Sh. Tonoyan, Y. Mamasakhlisov andV. MorozovDepartment of Molecular Physics, Yerevan State University,Yerevan 0025, 1 Alex Manoogian, Republic of Armenia

[email protected]

Biological macromolecules are mostly studied in aqueous sol-utions in the presence of other compounds (ligands) dis-solved in it (Badasyan et al., 2014; Asatryan, Tonoyan,Mirtavoosi, Mamasakhlisov, & Morozov, 2015; Vardevanyan,Antonyan, Parsadanyan, Torosyan, & Karapetian, 2016; DeCosta & Heemstra, 2013). It is meaningful theoretically toconsider biopolymer in a two-component solvent. Our paststudies in the frame of GMPC (Asatryan et al., 2015;Badasyan et al., 2014) resulted in the following expressionfor the concentration (c) dependence of helix-coil transitiontemperature (Tm):

cfðTm; fPLgÞ þ ð1 % cÞfðTm; fPSgÞ ¼ 0 (1)

where {PL} and {PS} are sets of parameters describing theinteraction of ligand and solvent with repeating unit corres-pondingly. Besides the variety of interaction schemes consid-ered, Tm(c) resulting from Equation (1) always remainsmonotonic. Meantime, several experimental studies(Vardevanyan, Antonyan, Parsadanyan, Torosyan, &Karapetian, 2016; De Costa & Heemstra, 2013) reported thenon-monotonic behavior. In order to reproduce such non-monotonicity, we had to separate the binding sites for eachof the solvent components in the model. To simplify, let usconsider the case, when both components (e.g. water andintercalating ligands in the case of DNA) stabilize the helicalstructure. The Hamiltonian of such model is comprised ofthree terms: one for the base model (Badasyan et al., 2014),and two terms, describing the interactions withtwo components.

Two solvents terms in the Hamiltonian are organized insuch a way to take into account the above-mentionedseparation of binding sites. As before (Asatryan et al.,2015; Badasyan et al., 2014), the model including solventsis reduced to the base model by the redefinition ofparameters. Repeating the same analysis as for Equation(1), we arrive at non-monotonic TmðcÞ " c2 dependence. Inthis particular case two stabilizing tendencies can result inthe appearance of the maximum on Tm(c). Thus our results


allow to explain the appearance of the extremum on thestability curve without invoking the existence of twoopposing mechanisms.

References

Asatryan, A., Tonoyan, S., Mirtavoosi, S., Mamasakhlisov, Y., & Morozov, V.(2015). 192 The helix-coil transition in two-component solvent in theframes of GMPC. Ligands effects on the characteristics of the transi-tion. Journal of Biomolecular Structure and Dynamics, 33(supp1),126–127.

Badasyan, A., Tonoyan, S., Giacometti, A., Podgornik, R., Parsegian, V.,Mamasakhlisov, Y., & Morozov, V. (2014). Unified description of solv-ent effects in the helix-coil transition. Physical Review E, 89(2), 022723.

De Costa, N., & Heemstra, J. (2013). Evaluating the effect of ionicstrength on duplex stability for PNA having negatively or positivelycharged side chains. PLoS One, 8(3), e58670

Vardevanyan, P., Antonyan, A., Parsadanyan, M., Torosyan, M., &Karapetian, A. (2016). Joint interaction of ethidium bromide andmethylene blue with DNA. The effect of ionic strength on bindingthermodynamic parameters. Journal of Biomolecular Structure andDynamics, 34(7), 1377–1382.

127. Coordinated actions of UvrA2during initiation of nucleotideexcision repair

Brandon C. Case and Manju M. HingoraniDepartment of Molecular Biology & Biochemistry, WesleyanUniversity, Middletown, CT 06459, USA

[email protected]

Nucleotide excision repair (NER) protects genomic DNA froma large number of chemically diverse lesions, includingnucleotide adducts and pyrimidine dimers. In bacteria, NER isinitiated by UvrA2 or UvrA2(B2) complex that scans DNA forchanges in structure/dynamics. Once the complex encoun-ters a potential damage site, UvrA2 dissociates, leaving UvrBto confirm lesion recognition and signal UvrC to nick thedamaged strand for removal (Kisker, Kuper, & van Houten,2013). Each monomer in the UvrA2 dimer has two ATPase sites(proximal and distal), both of which are required for NER andare implicated in lesion recognition and loading of UvrB ontothe DNA (Jaciuk, Nowak, Skowronek, Ta"nska, & Nowotny, 2011;Pakotiprapha, Samuels, Shen, Hu, & Jeruzalmi, 2012). Ourrecent study of the UvrA2 ATPase kinetic mechanism revealedthat the two sites exhibit asymmetric ATP binding and hydroly-sis, which is modulated differentially by undamaged and dam-aged DNA. The findings suggest that key steps in the ATPasemechanism may be coupled to changes in UvrA2 interactionswith DNA, ultimately leading to lesion recognition and UvrBloading (Case, Hartley, Osuga, Jeruzalmi, & Hingorani, 2019).We are now investigating the mechanochemical couplingbetween the ATPase reaction and other UvrA2 activities todetermine how the nucleotide-bound state of UvrA2 influencesits conformation and interactions with DNA. Preliminary resultsindicate the UvrA2-lesion complex adopts a distinct conform-ation in the presence of ATP that is not observed with ADP orwithout any nucleotide. Additionally, analysis of Walker A andB mutants to prevent ATP binding or hydrolysis, respectively, is

helping parse the role of each active site, and providing newinsights into how UvrA2 uses ATP to recognize lesions and initi-ate NER.

References

Case, B. C., Hartley, S., Osuga, M., Jeruzalmi, D., & Hingorani, M. M.(2019). The ATPase mechanism of UvrA2 reveals the distinct roles ofproximal and distal ATPase sites in nucleotide excision repair. NucleicAcids Research, under revision.

Jaciuk, M., Nowak, E., Skowronek, K., Ta"nska, A., & Nowotny, M. (2011).Structure of UvrA nucleotide excision repair protein in complexwith modified DNA. Nature Structural & Molecular Biology, 18(2),191–197.

Kisker, C., Kuper, J., & van Houten, B. (2013). Prokaryotic nucleotideexcision repair. Cold Spring Harbor Perspectives in Biology, 5(3),a012591.

Pakotiprapha, D., Samuels, M., Shen, K., Hu, J. H., & Jeruzalmi, D. (2012).Structure and mechanism of the UvrA-UvrB DNA damage sensor.Nature Structural & Molecular Biology, 19(3), 291–298.

128. Dynamics changes of CRISPR-Cas9 system induced by highfidelity mutations

Liangzhen Zheng and Yuguang MuSchool of Biological Sciences, Nanyang Technological University,637551, Singapore [email protected]

CRISPR-Cas9 as a powerful genome editing tool has widelybeen applied in biological fields. Ever since the discovery ofCRISPR-Cas9 as an adaptive immune system, it has beengradually modified to perform precise genome editing ineukaryotic cells by creating double-strand breaks. Thoughbeing robust and efficient, current CRISPR-Cas9 system facesa major flaw: the off-target effect, which has not been wellunderstood. Several Cas9 mutants show significant improve-ment, with very low off-target effect, however, relativelylower cleavage efficiency for on-target sequences as well. Inthis study, the dynamics of the wild-type Cas9 from S. pyo-genes and the high fidelity (HF) Cas9 mutant has beenexplored using molecular dynamics simulations. It turns outthat the mutations cause reduction of electrostatic interac-tions between Cas9 and R-loop. Consequently, the flexibilityof the tDNA/sgRNA heteroduplex is reduced, which mayexplain the reason of less tolerance of mismatches in theheteroduplex region. The mutations also affect the proteindynamics and the correlation networks among Cas9 domains.In mutant Cas9, weakened communications between twocatalytic domains, as well as the mutations induced slightopening of the conformation, may account for the lower on-target cleavage efficiency, and probably lower off-target aswell. These findings would facilitate more precise Cas9engineering in future.

Funding

This research has been supported by MOE (Tier 1 RG146/17).


129. Effect of concentration of ligandson DNA-biosensor average outputsignal and dispersion

Valeri B. Arakelyana, Marine A. Parsadanyanb andPoghos O. VardevanyanbaDepartment of Molecular Physics; bDepartment of Biophysics,Yerevan State University, Yerevan, Armenia

[email protected]

Nowadays, DNA-biosensors are intensively applied for reso-lution of actual bio-analytical and medical problems, such asidentification of biological material by primary sequence ofnucleotides, improvement of early diagnostic and treatmentmethods of oncological diseases, determination of pharma-cological preparations of anti-cancerous action (Kavita, 2017).Application of DNA-biosensors significantly decreases bothanalysis cost and time of its carrying out as compared totraditional medico-biological methods. It is connected to thefact that DNA-biosensors have small sizes and demand rele-vantly less amounts of the studying samples. Meanwhile, thedecrease of DNA-biosensor sizes inevitably results in bothfalling down of its output signal value and increasing of levelof the signal fluctuations. It should be mentioned that DNA-biosensor output signal fluctuations may be conditioned byarbitrary character of the binding of small molecules (ligands)in the solution with DNA duplexes on the biosensor as well.By this reason, the studies of the effect of ligands on averagevalue of DNA-biosensor output signal and its fluctuationsbecome actual and important. In this work in the frameworkof relativistic approach the dependences of DNA-biosensoroutput signal average value and its dispersion on ligand con-centration in the solution were theoretically calculated. Thecharacteristic peculiarities of DNA-biosensor output signalwere studied. It was shown that the output signal of DNA-biosensors monotonously increases with ligand concentrationenhancement in the solution (Arakelyan et al., 2006). It wasalso shown that the output signal relaxation time decreasesalong with ligand concentration increasing in the solution.Calculations showed that the dispersion dependence ofDNA-biosensor output signal on the ligand concentration inthe solution has a form of a curve with maxima. From the

beginning with concentration increasing of ligands in thesolution DNA-biosensor output signal dispersion increases,then passing through the maximum it decreases and tendsto zero at the further enhancement of ligand concentration.The obtained results can be used in the practice at prepar-ation of micro DNA-biosensors and analysis of the measure-ment data.

References

Arakelyan, V. B., Babayan, S. Y., Tairyan, V. I., Arakelyan, A. V.,Parsadanyan, M. A., & Vardevanyan, P. O. (2006). Kinetics of ligandbinding to nucleic acids. Journal of Biomolecular Structure andDynamics, 23(4), 479–484.

Kavita, V. (2017). DNA Biosensors – A Review. Journal of Bioengineeringand Biomedical Science, 7, 222. doi:10.4172/2155-9538.1000222

130. Expanding the structuraldiversity of DNA beyond thedouble helix

Betty Chu and Paul J. PaukstelisDepartment of Chemistry and Biochemistry, Center forBiomolecular Structure and Organization, University of Maryland,College Park, MD 20742, USA [email protected]

The iconic model of DNA is the Watson-Crick double helix,but it can form other types of structures. The extent of thestructural diversity of DNA is not well understood. We areinterested in discovering new types of DNA structurethrough the screening of a library of short DNA sequencesand subsequent structure determination for those that crys-tallize. From the screen, we aim to create a library of 3DDNA crystal structures and identify previously unknown DNAmotifs. We report two new crystal structures of single oligo-nucleotides that interact via noncanonical base pairing.d(CGTAAGGCG) forms a non-G-quadruplex fold-back struc-ture through both Watson-Crick and noncanonical interac-tions. The tetrameric assembly encloses a central cationbinding pocket and features a hexad base pairing arrange-ment through two C-G-G base triples. This is the first fold-

Figure 1.


back structure that forms a tetramer and is specific for diva-lent cations. We have also determined three variant sequen-ces that form the same structure, suggesting that there is alarge number of potential fold-back sequences in genomes(Chu, Zhang, Hwang, & Paukstelis, 2018). Fold-back struc-tures are biologically relevant since they have beenobserved in promoter regions of development genes (Miret al., 2017). d(CCAGGCTGCAA) features a Ba2þ-stabilizedG-quadruplex, which is flanked on either side by a basetriple formed through noncanonical interactions and ani-motif. This tetramer is the first structure of a hybrid DNAG-quadruplex/i-motif and demonstrates the possibility ofthe coexistence of G-quadruplexes and i-motifs in a singlestrand of DNA in genomes. The fold-back quadruplex andhybrid G-quadruplex/i-motif highlight the growing struc-tural diversity of DNA and suggest greater biological rolesfor non-duplex structures. These two structures demon-strate that DNA assemblies beyond the traditional doublehelix exist and suggest that DNA can form even morediverse structures (Figure 1).

Funding

This research has been supported by the NSF (CareerAward 1149665).

References

Chu, B., Zhang, D., Hwang, W., & Paukstelis, P. J. (2018). Crystal structureof a tetrameric DNA fold-back quadruplex. Journal of the AmericanChemical Society, 140(47), 16291–16298.

Mir, B., Serrano, I., Buitrago, D., Orozco, M., Escaja, N., & Gonzalez, C.(2017). Prevalent sequences in the human genome can form mini i-motif structures at physiological pH. Journal of the American ChemicalSociety, 139(40), 13985–13988.

131. Interaction of ethidium bromideand mitoxantrone with double-stranded poly(rA)-poly(rU)

Armen T. Karapetyana, Margarita A. Torosyana andMariam A. ShahinyanbaDepartment of Physics and Electrotechnics, Natioanl University ofArchitecture and Construction of Armenia, Yerevan, 0009,Armenia; bDepartment of Biophysics, Yerevan State University,Yerevan, 0025, Armenia [email protected]

The study of binding of the ligands ethidium bromide (EtBr)and mitoxantrone (MTX) with double-stranded (ds-) DNA (B-form) and ds-poly(rA)-poly(rU) (ds-RNA, A-form) has been car-ried out. The obtained results reveal that EtBr forms severaltypes of complexes with both ds-DNA and ds-poly(rA)-poly(rU) structures: by intercalation, semi-intercalation andelectrostatic modes simultaneously. Though, it was revealedthat EtBr shows practically the same affinity to ds-DNA andds-poly(rA)-poly(rU). MTX binds to ds-DNA and ds-RNA bysemi-intercalation and electrostatic modes and performs

some preference to DNA (to B-form). Our obtained data alsoindicate that MTX binds to DNA by analogous mechanism withmethylene blue (MB):—at relatively high ionic strengths andlow concentrations it semi-intercalates into ds-structure ofnucleic acid (NA), at high concentrations of the ligands—bindsto helix electrostatically from the outer side (Vardevanyan,Antonyan, Parsadanyan, Torosyan, & Karapetian, 2016). It wasalso revealed that at the joint binding of both ligands to NAand their low concentrations a competition between boundEtBr molecules and MTX does not emerge, meanwhile theirgeneral effect on stabilization of DNA ds-structures is less, thanat separate impacts. At high concentrations of both ligands inthe case of ds-DNA a competition between them emerges,though, the EtBr effect becomes prevailing. At the same timein the case of the joint interaction of both ligands withpoly(rA)-poly(rU) the main part of stabilization of ds-structurescorresponds to EtBr, which may be the result of MTX weakaffinity to ds-poly(rA)-poly(rU).

Thus, the experimental data indicate that despite the ver-satility of the main (semi-intercalation) binding mode, MTXinteracts with RNA much weaker, than with DNA. From thispoint of view, in the case of EtBr it was not revealed a pref-erence to DNA or RNA. It was shown that the EtBr bindingmodes to the NA does not depend on the absence or thepresence of MTX.

Reference

Vardevanyan, P. O., Antonyan, A. P., Parsadanyan, M. A., Torosyan, M. A.,& Karapetian, A. T. (2016). Joint interaction of ethidium bromide andmethylene blue with DNA. The effect of ionic strength on bindingthermodynamic parameters. Journal of Biomolecular Structure andDynamics, 34(7), 1377–1382. DOI: http://dx.doi.org/10.1080/07391102.2015.1079557.

132. Mismatched base pairs locallydistort DNA structure, leading toincreased DNA-binding bytranscription factor proteins

Ariel Afeka,b, Honglue Shic, Atul Rangaduraid,Hashim M Al-Hashimic,d and Raluca M Gordana,baCenter for Genomic and Computational Biology, Duke University,Durham, NC 27708, USA; bDepartment of Biostatistics andBioinformatics, Duke University, Durham, NC 27708, USA;cDepartment of Chemistry, Duke University, Durham, NC 27708,USA; dDepartment of Biochemistry, Duke University, Durham, NC27708, USA [email protected]

The local structure of genomic DNA can vary drastically fromthe ideal B-form double helix, and one cause for structuraldeformations is the pairing of non-complementary bases (i.e.,mis-paired bases, or mismatches). DNA mismatches are fre-quently formed by spontaneous chemical reactions (such asnucleotide deamination), replication errors, and geneticrecombination. Mismatches alter the local DNA structure(Figure 1a), which can affect interactions with DNA-binding


proteins, including regulatory transcription factors (TFs).Currently, very little is known about the effects of mis-matches on TF binding.

We present Saturation Mismatch Binding Assay (SaMBA),the first assay to characterize the effects of mismatches onTF-DNA binding in high throughput. For genomic sequencesof interest, SaMBA generates DNA duplexes containing allpossible single-base mismatches, and quantitatively assessesthe effects of the mismatches on TF-DNA interactions.

We applied SaMBA to measure binding of 21 TFs (cover-ing 14 structural families) to thousands of mismatchedsequences, and mapped the impact of mismatches on theseTFs. Remarkably, for all 21 TFs examined, introduction of mis-matches at certain positions resulted in significantlyincreased binding, with some mismatches creating high-affin-ity binding sites in nonspecific DNA and some convertingknown binding sites into ‘super-sites’ stronger than anycanonical Watson-Crick site.

Structural analyses of mismatches that increase TF bindingrevealed that these mismatches are oftentimes distorting thenaked DNA such that its structure becomes similar to that ofbound DNA sites (Figure 1(b)), thus explaining the increasedbinding measured in our assay. Our results reveal that theintrinsic energy cost of deforming the DNA structure is animportant, widespread layer of control in protein-DNA recog-nition. Furthermore, since several recent studies have shownthat bound TFs at damaged DNA are likely to be a significantcause for mutations (Reijns et al. 2015), characterizing thebinding preferences of TFs to mismatched DNA is an import-ant step toward understanding the mutational landscape inthe genome.

References

Reijns, M. A. M., Kemp, H., Ding, J., Marion de Proc"e, S., Jackson, A. P., &Taylor, M. S. (2015). Lagging-strand replication shapes the mutationallandscape of the genome. Nature, 518(7540), 502–506.

Rohs, R., Jin, X., West, S. M., Joshi, R., Honig, B., & Mann, R. S. (2010).Origins of specificity in protein-DNA recognition. Annual Review ofBiochemistry, 79, 233–269.

133. Modeling of protein–DNAbinding with a multi-module deeplearning framework

Tsu-Pei Chiu, Satyanarayan Rao, Ana Carolina DantasMachado and Remo Rohs

Quantitative and Computational Biology, Department of BiologicalSciences, University of Southern California, Los Angeles, CA, USA

[email protected]

Transcription factor (TF)-DNA binding is a fundamental compo-nent of gene regulatory processes, but how these proteins recog-nize their target sites in the genome is still not completelyunderstood. TFs can recognize their binding sites by having a sur-face physicochemically complementary to the physicochemicalsignature of DNA, forming a series of contacts between aminoacids and nucleotides. These contacts include direct hydrogenbonds, water-mediated hydrogen bonds, and hydrophobic con-tacts. In the last decade, several high-throughput technologieshave been developed for a better understanding of the TF-DNAbinding mechanisms by quantitatively measuring the bindingaffinities of a TF against thousands or even millions of differentDNA sequences in vitro. Relevant methods include high-through-put sequencing technologies, such as SELEX-seq, HT-SELEX orSMiLE-seq. These methods provide an alternative path to infer TF-DNA binding mechanisms without requiring time-consumingstructural biology experiments. Here, we present DeepRec (DeepRecognition for TF-DNA binding), a multi-module deep learningframework capable of building a precise predictive model for TF-DNA binding based on large-scale in vitro experimental data. Themethod integrates a forward perturbation-based interpretationapproach to highlight the important patterns for deciphering thebinding mechanisms. We demonstrate here applications of ourmethod to SELEX-seq data for human helix-loop-helix (bHLH) pro-tein MAX, the human myocyte enhancer factor-2B (MEF2B), andthe human tumor suppressor protein p53. We accurately pre-dicted DNA binding specificities and were able to unravel import-ant insights into the binding mechanisms.

134. Modeling physical analogsof DNA

Valentina N. Balashovaa, Ludmila V Yakushevichb andFarit K. ZakiryanovaaBashkir State University, Ufa, 450076, Russian Federation;bInstitute of Cell Biophysics RAS, Pushchino, 142290, RussianFederation [email protected]

The creation of mechanical, electronic and other physicalanalogs of living systems, including whole organisms, indi-vidual organs, cells, and even biological molecules, helps todeepen our knowledge of the nature of these systems. Inthis paper, we consider a possibility to create the mechanical

Figure 1. (a) DNA mismatches have non-canonical base-pair geometries and induce large structural. (b) An example of protein-induced DNA structural deformationthat could be mimicked by specific mismatches.


and electronic analogs of the DNA molecule (Figure 1), aswell as mathematical methods that allow to calculate theparameters of these analogs. The goal of this research is tofind the coefficients of the transformation that relates thestructural and dynamic parameters of DNA with similarparameters of the mechanical and electronic analogs of themolecule. The basis of the proposed approach is the ideas ofScott (1969) and of Englander, Kallenbach, Heeger,Krumhansl, and Litwin, (1980), supplemented by recentlyproposed mathematical model of the DNA torsion dynamics(Grinevich, Ryasik, & Yakushevich, 2015). They allowed us tofind (1) the transformation required, (2) the values of theparameters of the mechanical analog (pendulums mass m,pendulums length r, stiffness of springs K, the distancesbetween neighboring pendulums a), (3) the values of theelectronic circuit parameters (L, C, I0, d) and (4) the relation-ship between the parameters of the mechanical and elec-tronic analogues and the parameters of DNA (M, K b, R). Theobtained results show that the creation of the mechanicaland electronic analogs of the DNA molecule can be realizedand used to study various dynamic regimes that occur in themolecule, in particular, the modes associated with the emer-gence and spread of the transcription bubbles that play animportant role in the functioning of the DNA molecule.

References

Scott, A. C. (1969). A nonlinear Klein-Gordon equation. American Journalof Physics, 37(1), 52–61.

Englander, S. W., Kallenbach, N. R., Heeger, A. J., Krumhansl, J. A., &Litwin, S. (1980). Nature of the open state in long polynucleotide dou-ble helices: Possibility of soliton excitations. Proceedings of theNational Academy of Sciences of the United States of America, 77(12),7222–7226.

Grinevich, A. A., Ryasik, A. A., & Yakushevich, L. V. (2015). Trajectories ofDNA bubles. Chaos, Solitons & Fractals, 75, 62–75.

135. Modifications in the oxidativeand constructive metabolism of yeastYarrowia lipolytica caused bylimitation the growth by nitrogenor thiamine

Svetlana V. Kamzolova, Julia N. Lunina, Nadezhda N.Stepanova, Ramil K. Allayarov and Igor G. Morgunov

Federal Research Center Pushchino Center for Biological Researchof the Russian Academy of Sciences, G.K. Skryabin Institute ofBiochemistry and Physiology of Microorganisms RAS, Pushchino,Moscow region, 142290, Russia [email protected]

The study of oxidative and constructive metabolism underconditions of limitation of growth of microbial cultures isimportant from the point of view of the directional produc-tion of practically valuable metabolites, enzymes, biomasswith the special composition.

The object of the study was the yeast Yarrowia lipolytica,the uniqueness of which lies in the ability to synthesize signifi-cant quantities of organic acids, lipids and enzymes from awide range of carbon sources (Lazar, Liu, & Stephanopoulos,2018; Morgunov & Kamzolova, 2015; Zinjarde, 2014). Ethanolwas used as carbon source. Ethanol as a substrate for growthof producers of practically valuable compounds has severaladvantages over other substrates. It does not contain harmfulimpurities, it is well assimilated by yeast and dissolves in waterin any proportions. Since ethanol is used in the human diet,the products derived from it do not require additional purifica-tion from the residual substrate. As reported by Weusthuis,Aarts, and Sanders (2011) the several companies in the USAand Switzerland created the food products based on microbialbiomass produced from ethanol.

In the course of the research, it was established thatunder conditions of unlimited (exponential) growth of Y. lipo-lytica VKM Y-2373 the efficiency of ethanol oxidation attaineda maximum. Under those conditions the cells contained themaximum amount of protein, and the excretion of metabo-lites (citric, isocitric, a-ketoglutaric and other acids) did notoccur (Table 1). In other words, the metabolism of Y. lipoly-tica was relatively balanced.

On the contrary, under conditions of Y. lipolytica VKM Y-2373 growth limitation by nitrogen or thiamine, the complexof functional changes takes place (Table 1), of which themost significant modifications are following:

1. the orientation of biosynthetic processes shifts into thedirection of a sharp decline in protein synthesis;

2. the intensive excretion of cellular metabolites becameactive; the composition and quantity of the excretedorganic acids depend on the nature of the growth-limit-ing component: the limitation with a nitrogen stimu-lated the excretion of citric and isocitric acids (6.8 and

Figure 1.


11.2 g/L, respectively), while with a thiamine deficiencycells synthesize a-ketoglutaric acid (11.0 g/L).

3. the ratio of the enzymes responsible for the metabolismof the excreted products changes. Under unlimitedgrowth the activity of all enzymes involved in primaryoxidation of ethanol, Krebs and glyoxylate cycles wassufficiently high. Under conditions of nitrogen limitation,a high activities of citrate synthase and aconitate hydra-tase, necessary for the intensive synthesis of citric andisocitric acids, in distinction from the subsequentenzyme of Krebs cycle, NAD-isocitrate dehydrogenase,were observed. Hence, these acids formed in Krebs cyclecan be presumably excreted from the yeast cell ratherthan metabolized via the cycle. Similar data were previ-ously obtained for Y. lipolytica - producers of citric- andisocitric acids from biodiesel-derived waste (Morgunov &Kamzolova, 2015). In contrast, in the case of thiaminedeficiency, the metabolic flux is blocked at the level ofthe thiamine-dependent enzyme, 2-oxoglutaratedehydrogenase, and a-ketoglutaric acid is excreted intothe cultural medium rather than metabolized via thecycle. The continuous formation of excreted productswas ensured by the functioning of the glyoxylate cycle,with is confirmed by the high activities of citrate syn-thase, aconitate hydratase and isocitrate lyase whichoperate both in the glyoxylate cycle and in Krebs cycle.

References

Lazar, Z., Liu, N., & Stephanopoulos, G. (2018). Holistic approaches inlipid production by Yarrowia lipolytica. Trends in Biotechnology, 36(11),1157–1170. doi:10.1016/j.tibtech.2018.06.007

Morgunov, I. G., & Kamzolova, S. V. (2015). 166 Metabolism of biodiesel-derived waste in the yeast Yarrowia lipolytica—producers of citric-and isocitric acids. Journal of Biomolecular Structure and Dynamics,33(supp1), 108–109. doi:10.1080/07391102.2015.1032803

Weusthuis, R., Aarts, J., & Sanders, J. (2011). From biofuel to bio-product: Is bioethanol a suitable fermentation feedstock for syn-thesis of bulk chemicals? Biofuels, Bioproducts and Biorefining, 5(5),486–494.

Zinjarde, S. S. (2014). Food-related applications of Yarrowia lipolytica.Food Chemistry, 152, 1–10. doi:10.1016/j.foodchem.2013.11.117

136. Structure and hybridizationproperties of phosphorylguanidineoligonucleotides

Alexander A. Lomzov, Victor M. Golyshev,Evgeniya S. Dyudeeva, Maksim S. Kupryushkin andDmitriy V. PyshnyiInstitute of Chemical Biology and Fundamental Medicine SB RAS,Novosibirsk State University, Novosibirsk 630090, Russia

[email protected]

Oligonucleotide analogs have been a hot topic for the pastthree decades because of their present and potential thera-peutic applications. Recently, a new type of oligonucleotidederivatives with a modified backbone was developed—phos-phorylguanidine oligonucleotides (PGO) (Kuprushkin et al.,2014). PGOs are unique due to the possibility of synthesis viastandard automated phosphoroamidite protocol using basicset of unmodified phosphoroamidites, allowing to introducePG modification at any position of the oligomer sequence.The high potential of the PGOs was shown in biosensors, inbiological applications and in therapy (Lebedeva et al., 2015;Dmitrienko et al., 2016) (Figure 1).

In this work, we studied the solution structure of PGOsand their complementary complexes with unmodified DNAor RNA as well as thermal stability of these complexes underconditions of different ionic strength. There were no signifi-cant differences between the CD spectra of unmodified oli-gonucleotides and PGOs as well as between spectra ofunmodified duplexes and duplexes with one fully modifiedstrand, indicating that PG modification insignificantly disturbsthe structure of modified oligonucleotides and their duplexeswith native DNA or RNA.

At high ionic strength (1M NaCl), the thermal stability ofPGO containing duplexes decreases with each added PGmodification. At low ionic strength (<100mM NaCl), com-plexes with one fully modified strand are more stable thanthe native ones. Moreover, a fully modified PGO formedhighly stable duplexes with DNA or RNA even in deionized

Table 1. The effect of limiting factors on the growth and product synthesis inY. lipolytica VKM Y-2373.

ParametersUnlimitedgrowth

Limitation of growth by:

Nitrogen(63mg/L)

Thiamine(0.3 mg/L)

Protein (%) 50.75 21.2 27.50Lipids (%) 10.12 10.30 12.37Citric acid (g/L) 0 6.8 0.50Isocitric acid (g/L) 0 11.2 0.25a-Ketoglutaric acid (g/L) 0 0 11.00Other organic acids (%) 0 3.5 10.0Enzyme activite (U/mg protein)Alcohol dehydrogenase 0.07 0.06 0.05Aldehyde dehydrogenase 0.04 0.03 0.02Citrate synthase 1.71 1.80 1.65Aconitate hydratase 0.39 0.40 0.38NAD-isocitrate dehydrogenase 0.11 0.020 0.10NADP-isocitrate dehydrogenase 0.14 0.080 0.090Oxoglutarate dehydrogenase 0.11 0.070 0.010Isocitrate lyase 0.036 0.060 0.060

Figure 1. Structure of the phosphorylguanidine modification.


water. PGOs have sequence dependent hybridization proper-ties and can be used for single mismatch discrimination likeother native or modified oligonucleotides .

Funding

This research has been supported by the Russian ScienceFoundation (grant no. 18-14-00357).

References

Dmitrienko, E., Naumova, O., Fomin, B., Kupryushkin, M., Volkova, A.,Amirkhanov, N., … Pyshnyi, D. (2016). Surface modification of SOI-FET sensors for label-free and specific detection of short RNA analyte.Nanomedicine (London), 11(16), 2073–2082.

Kupryushkin, M. S., Pyshnyi, D. V., & Stetsenko, D. A. (2014). Phosphorylguanidines: A new type of nucleic acid analogues. Acta Naturae, 6(4),116–118.

Lebedeva, N. A., Anarbaev, R. O., Kupryushkin, M. S., Rechkunova, N. I.,Pyshnyi, D. V., Stetsenko, D. A., & Lavrik, O. I. (2015). Design of a newfluorescent oligonucleotide-based assay for a highly specific real-timedetection of apurinic/apyrimidinic site cleavage by tyrosyl-DNAphosphodiesterase 1. Bioconjugate Chemistry, 26(10), 2046–2053.

137. Study of three-componentsystem, consisted of DNA complexeswith ligands of different type

Ara P. Antonyan, Marine A. Parsadanyan, Narek H.Shahnazaryan and Poghos O. VardevanyanDepartment of Biophysics, Yerevan State University, Yerevan,Armenia [email protected]

The joint binding of intercalators EtBr and AO, intercalatorEtBr and semi-intercalator MB, intercalator EtBr and groove-binding compound Hoechst 33258 (H33258) with DNA hasbeen studied using UV melting method at ionic strengths ofthe solution 2 and 20mM. The values of melting temperature(Tm) and melting interval width (DT) of DNA-ligand com-plexes were obtained and based on them the values ofchanges of these parameters—(1/Tm) and (DT/Tm

2) on ligandconcentrations were determined. The obtained results indi-cate that at the joint binding of two different ligands withDNA, the solution ionic strength has a determining effect ofon their binding modes. Meanwhile, the joint interaction ofintercalator and non-intercalator or two intercalators withDNA is not a simple sum of their separate binding.Moreover, at their joint interaction with DNA a competitionbetween them emerges. These results also indicate that thejoint interaction of two different multimodal ligands withDNA is not a result of binding by at least two modes of onetype of ligand (Karapetian, Grigoryan, Mamasakhlisov,Minasyants, & Vardevanyan 2016).

Results of the experimental studies of DNA complexeswith two different ligands reveal that the immediate bindingof any type of the ligand with DNA leaves a separate effecton its thermodynamic characteristics, though the jointimpact of two (and, probably, more) different ligands is not

a simple sum of such influences. Moreover, in the certaindegree, the joint effect of two ligands on thermodynamiccharacteristics of DNA helix-coil transition is possible to con-sider as the effect of ‘generalized’ ligand, which shows intrin-sic properties for the latter (Nafisi, Saboury, Keramat, Neault,& Tajmir-Riahi, 2007). It is also obvious that the long polymerchain of DNA acts as one integer, since even at non-relevantconcentrations of two different ligands, when the regionsbetween these compounds outlying from each other arefilled by molecules, it behaves not as in the case of separatebinding at the same concentrations. This fact may elucidatesome questions, connected to DNA functioning regulation(initiation, inhibition, expression of genes) in cell.Nonetheless, the obtained data do not put in a claim forabsoluteness, since it cannot be excluded the additive effectof two different ligands on parameters of DNA conform-ational transitions, which may also have a certain biologicalvalue for DNA functioning regulation.

References

Karapetian, A. T., Grigoryan, Z. A., Mamasakhlisov, Y. S., Minasyants,M. V., & Vardevanyan, P. O. (2016). Theoretical treatment of helix-coiltransition of complexes DNA with two different ligands having differ-ent binding parameters. Journal of Biomolecular Structure andDynamics, 34(1), 201–202. doi: 10.1080/07391102.2015.1010584.

Nafisi, S., Saboury, A. A., Keramat, N., Neault, J.-F., & Tajmir-Riahi, H.-A.(2007). Stability and structural features of DNA intercalation with eth-idium bromide, acridine orange and methylene blue. Journal ofMolecular Structure, 827(1-3), 35–43.

138. The effect of porphyrinsconfiguration on the hydrodynamicbehavior of DNA solutions

Vigen G. Barkhudaryan and Gayane V. AnanyanDepartment of Molecular Physics, Yerevan State University, 1 AlexManoogian St. 0025, Yerevan, Armenia [email protected]

The present work summarizes possibilities of a viscometrymethod for studying the conformational changes of DNA atinteraction with porphyrins. For elucidating the influence ofporphyrin structure by varying the metal center, peripheralsubstituents and their positions in pyridylic ring on bindingmode and intensity of interaction with DNA water solublecationic meso-tetra-(3N- and 4N-hydroxyethylpyridyl)(Barkhudaryan & Ananyan, 2014, 2016), (3N- and 4N-allylpyr-idyl) (Barkhudaryan & Ananyan, 2017, 2018) porphyrins andtheir metal complexes with Cu and Co were studied via visc-ometry and UV–vis absorption spectroscopy. It was shownthat planar porphyrins interact with DNA by intercalationmode (Figure 1a, b), excepting CuTAllPyP4 which interactswith DNA by partial intercalation mod (Barkhudaryan &Ananyan, 2018). Moreover, the planar porphyrins with allyl-pyridyl peripheral substituents interact with DNA consider-ably more intensively than with hydroxyethylpyridylsubstituents. Comparison of different locations of peripheral


substituents on pyridylic rings leads to the conclusion thatthe H2THOEtPyP3 and H2TALPyP3 and their metallocom-plexes bind to DNA more preferably than H2THOEtPyP4,H2TALPyP4 and their metallocomplexes. This fact can beexplained by the preferred arrangement of 3N-porphyrins onthe DNA helix compared to 4N-porphyrins. The change ofchemical structure of peripheral radicals and their positionson pyridylic ring of porphyrins does not absolutely affect onthe mechanism of interaction of outside binders such as Co-porphyrins (Figure 1c). The viscometry and spectrophotomet-ric measurements are in good agreement (Barkhudaryan &Ananyan, 2014, 2016, 2017, 2018).

References

Barkhudaryan, V. G., Ananyan, G. V., Dalyan, Y. B., & Haroutiun, S. G.(2014). Development of viscometric methods for studying the inter-action of various porphyrins with DNA. Part I. Journal of Porphyrinsand Phthalocyanines, 18(07), 594–599.

Barkhudaryan, V. G., & Ananyan, G. V. (2016). Development of viscomet-ric methods for studying the interaction of various porphyrins withDNA. Part II. Journal of Porphyrins and Phthalocyanines, 20(07),766–772.

Barkhudaryan, V. G., & Ananyan, G. V. (2017). Development of viscomet-ric methods for studying the interaction of various porphyrins withDNA. Part III. Journal of Porphyrins and Phthalocyanines, 21(02),110–115.

Barkhudaryan, V. G., & Ananyan, G. V. (2018). Development of viscomet-ric methods for studying the interaction of various porphyrins withDNA. Part IV. Journal of Porphyrins and Phthalocyanines, 22(11),1022–1029.

139. The influence of CuTOEPyP4porphyrin on DNA damage inducedby high energy electronbeam radiation

L.R. Aloyan, Y.B. Dalyan and S.G HaroutiunianYerevan State University, Al. Manoogian 1, Yerevan 0025, Armenia

[email protected]

The study and understanding of the mechanisms of radiationcausing damage to DNA is one of the important problems inthe development of new cancer therapies and effective radio-sensitizers. The fast electrons directly ionize the DNA molecule,causing damage. These include single-strand breaks and dou-ble-strand breaks, DNA–DNA or DNA–protein cross-links. Theusage of the porphyrin-based Photodynamic Therapy (PDT) inconjunction with electron beam radiation therapy can be oneof the effective treatment methods for cancer. Porphyrins haveattracted the attention of researchers globally for applicationas photosensitizing agents in medicine. However, studies ofthe radiation damage at the molecular level in the presence ofsuch PDT agents and anticancer drugs like porphyrins are stillnecessary. The goal of our investigations was to examine DNAdamage in the presence of different amounts of Cu containingwater soluble cationic meso-tetra-(4N-oxyethylpyridyl) porphy-rin induced by 3 and 4MeV electron radiation depending onradiation doses in vitro. The samples with different relativeconcentrations of porphyrins per base pair were irradiated bythe electron beam. After the irradiation of samples, the meltingcurves (the dependence of percentage of denaturized DNA ontemperature) of investigated complexes were obtained. As themelting temperature of DNA is sensitive to double helix stabil-ity, it can be used as an indicator of strand breaks of DNA mol-ecules after radiation. Our results indicate that at the radiationdose 1Gy when the relative concentration of porphyrinsequals 0.01 and 0.02 per DNA base pair, the radiation effect onthe DNA structure is higher than the stabilizing effect of por-phyrins. At the higher value of the relative concentration ofporphyrins (0.04 and 0.06) the DNA stabilizing effect is moresignificant than DNA damage produced by an electron beam.

On the contrary at the radiation dose equal to 2Gy theradiation effect exceeds the porphyrins stabilizing effect onDNA at all examined relative concentrations.

Acknowledgments

The experimental studies were conducted within the framework of RAMES Committee of Science Projects no. 18T-1F055.

Figure 1.


References

Ananyan, G., Avetisyan, A., Aloyan, L., & Dalyan, Y. (2011). The stability ofDNA-porphyrin complexes in the presence of Mn(II) ions. BiophysicalChemistry, 156(1), 96–101.

Dalyan, Y. B., Haroutiunian, S. G., Ananyan, G. V., Vardanyan, V. I.,Yu.Lando, D., Madakyan, V. N., … Benight, A. S. (2001). Interaction ofmeso-tetra-(4-N-oxyethylpyridyl) porphyrin, its 3-N analog and theirmetallocomplexes with duplex DNA. Journal of Biomolecular Structureand Dynamics, 18(5), 677–687. [10.1080/07391102.2001.10506698]

O’Connor, A. E., Gallagher, W. M., & Byrne, A. T. (2009). Porphyrin andnonporphyrin photosensitizers in oncology: Preclinical and clinicaladvances in photodynamic therapy. Photochemistry and Photobiology,85(5), 1053

Swiderek, P. (2006). Fundamental processes in radiation damage of DNA.Angewandte Chemie International Edition, 45(25), 4056.

Vrouenraets, M. B., Visser, G. W., Snow, G. B., & van Dongen, G. A. (2003).Basic principles, applications in oncology and improved selectivity ofphotodynamic therapy.. Anticancer Research, 23(1B), 505

Wilson, B. C. (2002). Photodynamic therapy for cancer: Principles.Canadian Journal of Gastroenterology ¼ Journal Canadien deGastroenterologie, 16(6), 393

140. Molecular alterations in DNAunder UV-irradiation

Gayane V. Ananyan, Nelli H. Karapetyan andVigen G. BarkhudaryanDepartment of Molecular Physics, Yerevan State University, 1 AlexManoogian St. 0025, Yerevan, Armenia [email protected]

The ionizing radiation of matter produces a large number ofions, radicals, which can subsequently cause both physicaland chemical modification in the biological media. Moreoverthe irradiation can induce strand break formation in double-stranded supercoiled DNA.

In present work the influence of UV-irradiation on thestructural transformations of DNA in buffer solutions wasstudied using UV-absorption spectroscopy and viscometry.Detection of DNA structural changes such as singlestranded and double stranded breaks under UV-irradiationwere characterized by their melting curves, in which tem-perature-dependent spectral measurements were carriedout in buffered solutions for different exposure time/doseof irradiation. The changes of melting temperature (Tm) andinterval of helix-coil transition (DT) at different exposuretimes indicate damages to the secondary structure of DNA(Karapetyan, Torosyan, Malakyan, Bajinyan, & Haroutiunian,2016). The decreasing of melting temperature testifies toDNA destabilization, which could occur due to base modifica-tions, strand breaks or breaks of hydrogen bonds betweenthe DNA strands during irradiation. The increases of damagedsites create favorable conditions for helix-coil transition.Moreover, an increase in DT (degree of heterogeneity) alsoindicates the presence of defective sites. The relative viscosityof the UV-irradiated DNA solution decreased with increasingirradiation time, which indicates a reduction of macromole-cules size.

Studies have demonstrated that viscometry and spectro-photometric measurements are in good agreement.

References

Karapetyan, N. H., Torosyan, A. L., Malakyan, M. H., Bajinyan, S. A., &Haroutiunian, S. G. (2016). Investigation of irradiated rats DNA in thepresence of Cu(II) chelates of amino acids Schiff bases. Journal ofBiomolecular Structure and Dynamics, 34(1), 177–183.

141. Changes in the stability andconductivity of BLM in the presenceof zinc oxide nanoparticles

Anahit L. Torosyan, Gayane V. Ananyan andValeri B. ArakelyanDepartment of Molecular Physics, Yerevan State University, 1 AlexManoogian St. 0025, Yerevan, Armenia [email protected]

Currently, nanoparticles are intensively implanted in almostall areas of scientific and practical human activity, althoughthere are fears that they may damage biological systems(Limbach et al, 2005). The effect of nanoparticles beginningwith their primary interaction with biological membranes.However, since biological membranes display a very complexcomposition in terms of lipids and proteins, it seems appro-priate to investigate the effect of nanoparticles on a bilayerlipid membrane (BLM), which mimics well the lipid bilayer ofa biological membrane. This work is devoted to the study ofthe effect of zinc oxide nanoparticles (ZnO), with an averagesize of 50 nm, on the stability and conductivity of BLM. Theexperiments were performed on BLM obtained from a mix-ture of 1,2-dipalmitoyl-sn-glycero-3-[phospho-1-serine](DPPS)and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) ofthe company Avanti Polar Lipids in a ratio 1:1. Voltage, inthe range of 0.20–0.55 V, was applied to the BLM usingchlorine-silver electrodes connected to the ADC and con-trolled by a computer. Experimental points were obtaineddepicting the dependence of the average BLM lifetime onthe potential in the absence and presence of zinc oxidenanoparticles in solution. The parameters characterizing theBLM stability were determined from fitting the experimentaldata with a theoretical curve for the dependence of the aver-age lifetime of the BLM on the potential (Pastushenko,Chizmadzev, & Arakelyan, 1979). This is the BLM tension, thelinear tension of the pore edge in the BLM, as well as theparameter which is the product of the number of pores onthe BLM and the pore diffusion coefficient in radius space. Itwas shown that ZnO nanoparticles increase BLM stability inan electric field. With an increase in the concentration ofnanoparticles, the stability of BLM also increases. It wasshown that ZnO nanoparticles increase the value of the lin-ear tension coefficient of the pore edge, which is formed inBLM, which also leads to an increase in the stability of BLMin an electric field. It was also shown that ZnO nanoparticleslead to a decrease in the conductivity of BLM.


References

Limbach, L. K., Li, Y., Grass, R. N., Brunner, T. J., Hintermann, M. A.,Muller, M. … , Stark, W.J. (2005). Oxide Nanoparticle Uptake inHuman Lung Fibroblasts: Effects of Particle Size, Agglomeration, andDiffusion at Low Concentrations. Environmental Science & Technology,v39(23), 9370–9376.

Pastushenko, V. F., Chizmadzev, Y. A., & Arakelyan, V. B. (1979). ElectricBreakdown of Bilayer Lipid Membranes II. Calculation of the mem-brane lifetime in the steady-state diffusion approximation.Bioelectrochemistry and Bioenergetics, v. 6 p, 37–52.

142. Influence of low-intensitymillimeter waves on lipidperoxidation and antioxidantenzymes activity in male Wistar rats

Gayane H. Poghosyan, Marieta S. Mikaelyan andAnahit V. NerkararyanDepartment of Biophysics, Yerevan State University, Yerevan,Armenia [email protected]

Electromagnetic waves of millimeter range (MM EMW) corres-pond to the extremely-high-frequency (EHF) electromagneticirradiation (EMI) band (f ¼ 30–300GHz) always exist in sur-rounding environment and intensively influence living organ-isms. It has been suggested that initial cellular event affectedby exposure to EMI might be the increase of free radicallevel, which may enhance lipid peroxidation (LPO) and anti-oxidant enzymes activity (Ramundo-Orlando, 2010). Theinvestigation concerned with the effect of low-intensityMMW on whole body exposure of male Wistar rats. Twentyrats were divided into two groups: sham-exposed (control)and experimental (10 animals each). Animals were exposedwith 50.3 GHz frequency EMI (power flux density 64 mWt/cm2) for 1 h/day, for 5 days (day after day). As a source ofmonochromatic EMI EHF generator G4-141 type with work-ing interval of 37.50–53.57 GHz (State Scientific-ProductionEnterprise Istok’, Russia) was used. The whole body specificabsorption rate (SAR) was 0.05W/kg. Exposure took place ina ventilated plexiglas cage, where rats could move insidegiven space. After completion of each exposure period, ratswere sacrificed to analyze malondialdehyde (MDA)-rate andenzyme activity (catalase and glutathione peroxidase) inbrain, liver, heart and skeletal muscle. Our findings indicate asignificant increase (p< 0.05) in level of MDA and CAT activ-ity in brain of EMI-exposed group of animals as compared tosham-exposed, depending on EMI exposure duration. At thesame time, we recorded slightly elevated MDA-rate in experi-mental animals liver in comparison with the control group.The MDA values concerning heart and skeletal musclesremained at the same levels in the EMI- exposed rats anddid not differ significantly from control. Data show a signifi-cant decrease of GPX activity in experimental rats brain(p< 0.001) and liver (p< 0.05), whereas CAT activity signifi-cantly increased in the same organs compared to control.

Studied indexes may indicate possible health implications ofsuch exposure and should be taken into account.

References

Ramundo-Orlando, A. (2010). Effects of millimeter waves on cell mem-brane—a brief review. Journal of Infrared, Millimeter, and TerahertzWaves, 31(12), 1400–1411.

143. Nitrogen limitation triggers lipidsynthesis of Torulaspora globose onvarious carbon substrates

Nadezhda N. Stepanova, Svetlana V. Kamzolova andIgor G. MorgunovFederal Research Center Pushchino Center for Biological Researchof the Russian Academy of Sciences, G.K. Skryabin Institute ofBiochemistry and Physiology of Microorganisms RAS, Pushchino,Moscow region 142290, Russia [email protected]

It is well known that the nature of the growth-limiting com-ponent has a significant effect on lipid synthesis in microor-ganisms with a typical diphase process. The limitation ofyeast growth by mineral components of nutrition medium(for example, nitrogen) stimulates the intensive storage oflipids by the lipid-producer yeast, while with a carbon sourcedeficiency, microorganisms synthesize a negligible amount oflipids (Dourou et al., 2018; Lazar et al., 2018).

The purpose of this work was to study the effect of thenature of the growth-limiting component on biomass com-position and lipid synthesis of yeast Torulaspora globoseVKPMY-953, cultivated on ethanol or glucose.

The selected strain T. globose VKPM Y-953 has a uniquefeature—the ability to intensively synthesize lipids in parallelwith the growth of the culture, unlike the classical producersof lipids, in which intensive synthesis of lipids occurs afterthe completion of active growth.

As seen from Table 1, in the medium with ethanol as acarbon source under conditions of limitation of T. globosegrowth by nitrogen, a lipid synthesis was higher (43.8% ofthe biomass) than with an ethanol deficiency (17.8% of thebiomass). Conversely, with ethanol deficiency, the biomasswas characterized by a higher protein content (23.4% of bio-mass) and a biomass yield from the consumed substrate (YX/S) (54.4%) than with nitrogen limitation (16.7% of biomassand 31.1%, respectively); under these conditions, the lipidyield (YL/S) was higher by 27% than with a nitrogen defi-ciency. It is considered that lipids synthesized by microorgan-isms under deficiency of carbon substrate perform mainly afunctional role in metabolism (take part in transport proc-esses, regulate the activity of enzymatic systems, etc.). Thepurpose of growth-coupled lipid synthesis in T. globose is stillunknown. It is possible that the studied strain is character-ized by a higher proportion of functional lipids than othertypes of yeast.


The specific rates of lipid and protein synthesis were cal-culated per the unit of a lipid-free biomass fraction. The datapresented in Table 1 show that the specific rate of lipid syn-thesis exceeded the specific rate of protein synthesis by 2times with a nitrogen limitation and it was reduced by 1.7times with an ethanol deficiency. Thus, the lipid accumula-tion in the cells of T. globose depends on the ratio betweenthe rates of lipid and protein synthesis.

As seen from Table 1, the nature of the growth-limitingcomponent had no significant effect on the fatty acid (FA)composition of lipids in T. globose. The predominant fattyacids included the unsaturated acids —oleic acid (C18:1)(41.5–51.5% of total FA), palmitoleic acid (C16:1)(26.2–29.2% of total FA) and palmitic acid (C16)(14.5–15.5% of total FA).

Lipogenesis of T. globose cultivated on glucose had thedistinctive features: the lipid accumulation was reduced by2.7 times in comparison with cells, grown on ethanol; themaximum amount of lipids (16% of the biomass) was synthe-sized with a nitrogen deficiency, while with a glucose defi-ciency, the biomass yield from the consumed substrate (YX/S)was maximum (21.8%); the fraction of oleic acid (C18:1) in thelipids was increased up to 63% (of the total amount of FA).

Thus, nitrogen limitation triggers lipid synthesis of T. glo-bose on various carbon substrates.

Funding

The reported research was funded by RFBR (project @ 18-38-00794_mol_a).

References

Dourou, M., Aggeli, D., Papanikolaou, S., & Aggelis, G. (2018). Criticalsteps in carbon metabolism affecting lipid accumulation and theirregulation in oleaginous microorganisms. Applied Microbiology andBiotechnology, 102(6), 2509–2523. doi: 10.1007/s00253-018-8813-z.

Lazar, Z., Liu, N., & Stephanopoulos, G. (2018). Holistic approaches inlipid production by Yarrowia lipolytica. Trends in Biotechnology, 36(11),1157–1170. doi:10.1016/j.tibtech.2018.06.007

144. Zinc supplementation stimulateslipid production in Torulasporaglobose yeast

Svetlana V. Kamzolovaa, Nadezda N. Stepanovaa,Grigorii I. Morgunovb, Ramil K. Allayarova andIgor G. MorgunovaaFederal Research Center Pushchino Center for Biological Researchof the Russian Academy of Sciences, G.K. Skryabin Institute ofBiochemistry and Physiology of Microorganisms RAS, Pushchino,Moscow region, 142290, Russia; bPeoples’ Friendship University ofRussia (RUDN University), Miklukho-Maklaya str. 6, Moscow, P.O.Box 117198, Russia [email protected]

The practical importance of microbial lipids causes great inter-est of researchers to the problem of their synthesis and regula-tion. The experimental approaches in which the cell growthand the direction of various biosynthetic processes can be con-trolled are well known. The limitation of cell growth by nutri-tion elements is one of the ways to shift metabolism intodirectional synthesis of lipids (Athenaki et al., 2018).

At present, the ethanol is considered to be a promising car-bon source in various biotechnological processes, because itcan be produced from sugarcane, beet, corn, lignocelluloseand other renewable materials. It does not contain harmfulimpurities; it is well assimilated by yeast and dissolves in waterin any proportions. Several companies in the US andSwitzerland have created food products based on microbialbiomass produced from ethanol (Weusthuis et al., 2011). Itshould be noted that the large-scale production of lipids fromethanol is still limited by the lack of basic knowledge about fer-mentation conditions conducive to product overproduction.

Analysis of the literature data shows the exceptional role ofzinc in the regulation of cell metabolism and physiology.Besides acting as a cofactor for many enzymes (dehydrogen-ases, aldolases, polymerases and proteases) (Dedyukhina &Eroshin, 1991; Tomaszewska et al., 2014; Kamzolova et al.,2018) zinc is also required for the structural stability of zinc fin-ger proteins, many of which exert important controls on cellu-lar metabolic processes (Hamed & Arya, 2016).

The purpose of this work was to study the effect of vari-ous zinc concentrations on growth and lipid synthesis ofyeast Torulaspora globosa VKPMY-953 using a chemostateregimen (D ¼ 0.05 h% 1).

As seen from Table 1, the limitation of the growth of T. glo-bosa by zinc (0.001mg/L) sharply reduced the biomass yield(YX/S) (in 2.7 times), lipid content (in 3 times) and lipid yield (in3.6 times) as compare to high Zn concentration (2.8mg/L). Atthe same time, the protein content was maximum (37.96%from dry biomass) under condition of Zn deficiency.

As seen from Table 1, the lipids contained $14–$18 acidswith a prevalence of palmitic (C16:0), palmitoleic (C16:1) andoleic (C18:1) acids.

In this study, we focused on the palmitoleic acid (C16:1),which is considered as a novel lipokine (Athenaki et al., 2018).As seen from Table 1, a large amount of palmitoleic acid(37.5–41.7% of lipids) was synthesized at zinc concentration inthe range from 0.1 to 2.8mg/L.

Table 1. The effect of the nature of the growth-limiting component onphysiological indices of T. globose.

ParametersNitrogenlimitation

Ethanollimitation

Lipids (% of biomass) 43.8 27.8Protein ((% of biomass) 16.7 23.4Specific rate of lipid synthesis (h% 1) 0.04 0.03Specific rate of protein synthesis (h-1) 0.02 0.05Biomass yield by mass (YX/S) (g/g) 31.1 54.4Lipid yield by mass (YL/S) (g/g) 13.6 17.3Fatty acids (% of lipids)C14 0.2 0.1C14:1 Trace TraceC16 14.5 15.5C16:1 26.2 29.2C18 2.3 1.3C18:1 51.5 41.5$18:2 5.3 12.4Unsaturated fatty acids (%) 83.0 83.0

The data represent the means of four measurements. Standard deviation didnot exceed 10%.


The activity of D-9 desaturase involved in the conversionof palmitic into palmitoleic acid was evaluated by measuringthe ratio $16:1/$16. In our experiments, the ratio $16:1/$16

completely correlated with a change in the palmitoleic acidcontent of lipids (Table 1).

Funding

The reported research was funded by RFBR (project no. 18-38-00794_mol_a).

References

Athenaki, M., Gardeli, C., Diamantopoulou, P., Tchakouteu, S. S., Sarris, D.,Philippoussis, A., & Papanikolaou, S. (2018). Lipids from yeasts andfungi: Physiology, production and analytical considerations. Journal ofApplied Microbiology, 124(2), 336–367. doi: 10.1111/jam.13633.

Dedyukhina, E. G., & Eroshin, V. K. (1991). Essential metal ions in the con-trol of microbial metabolism. Process Biochemistry, 26(1), 31–37.10.1139/cjm-2018-0050

Hamed, M. Y., & Arya, G. (2016). Zinc finger protein binding to DNA: Anenergy perspective using molecular dynamics simulation and freeenergy calculations on mutants of both zinc finger domains and theirspecific DNA bases. Journal of Biomolecular Structure and Dynamics,34(5), 919–934. doi: 10.1080/07391102.2015.1068224.

Kamzolova, S. V., Shamin, R. V., Stepanova, N. N., Morgunov, G. I., Lunina,J. N., Allayarov, R. K., … Morgunov, I. G. (2018). Fermentation condi-tions and media optimization for isocitric acid production from etha-nol by Yarrowia lipolytica. BioMed Research International, 2543210,2018, 1. doi:10.1155/2018/2543210

Tomaszewska, L., Rymowicz, W., & Rywi"nska, A. (2014). Mineral sup-plementation increases erythrose reductase activity in erythritolbiosynthesis from glycerol by Yarrowia lipolytica. AppliedBiochemistry and Biotechnology, 172(6), 3069–3078. doi: 10.1007/s12010-014-0745-1.

Weusthuis, R., Aarts, J., & Sanders, J. (2011). From biofuel to bioproduct:Is bioethanol a suitable fermentation feedstock for synthesis of bulkchemicals? Biofuels, Bioproducts and Biorefining, 5(5), 486–494.

Zhao, X. Q., & Bai, F. W. (2012). Zinc and yeast stress tolerance:Micronutrient plays a big role. Journal of Biotechnology, 158(4),176–183. doi: 10.1016/j.jbiotec.2011.06.038.

Table 1. Effect of zinc amount on biomass yield and the amount of lipids inT. globosa.

Parameters

Zinc content (mg/L)

0.001 0.1 0.28 2.8

Biomass yield by mass (YX/S) (g/g) 0.21 0.57 0.57 0.56Lipids (% of biomass) 9.2 20.0 26.4 27.8Lipid yield by mass (YL/S) (g/g) 0.048 0.112 0.141 0.172Protein (% of biomass) 37.96 26.82 20.23 23.03Fatty acids (% of lipids)C14 0.8 2.1 3.3 2.5C14:1 0.7 0.6 1.0 0.4C16 16.5 18.6 23.8 24.6C16:1 29.2 41.7 40.7 37.5C18 3.3 2.1 1.2 2.5C18:1 42.2 24.9 30.0 32.5$18:2 7.3 0 0 0$16:1/C16 (D-9 desaturase) 1.87 2.24 1.71 1.52


Author Index

Abolghasemi, M. 30Abrahms!en, L. 39Afek, A. 80Agajanian, S. 24Alexander, S. P. 34Alexandrov, B. 47Al-Hashimi, H. M. 80Allayarov, R. K. 82, 88Aloyan, L.R. 85Alvareda Migliaro, E. M. 18Ananyan, G. V. 84, 86Andrianov, A. M. 22, 23Antonyan, A. P. 75, 77, 84Apukhtina, V. S. 76Arakelyan, V. B. 79, 86Arjmand, B. 36Arora, R. 29, 33Artsruni, I. G. 56, 71Artsruni, I. 52, 57Asatryan, A. L. 56, 71Asatryan, A. 52, 57, 77Astl, L. 24Avagyan, G. A. 21Avison, M. B. 16Bairagya, H. R. 66Balashova, V. N. 81Bandyopadhyay, D. 64Ban!a"s, P. 48, 50Barbar, E. 9Barkhudaryan, V. G. 84, 86Barlow, J. 61Barton, J. K. 63Barton, J. 63Basu, A. 7Beattie, N. R. 8Belfort, G. 19Bellini, D. 14Best, R. B. 50Beveridge, D. L 6Bhadra, K. 21Bhat, R. 17Bhatnagar, A. 64Bhattacherjee, A. 75Bischoff, N. 24Bishop, T. C. 55Biswas, S. 44Blacklock, K. 24Bondos, S. E. 10

Brehove, M. S. 44Buragohain, M. 13Bussi, G. 50Caetano-Anoll!es, G. 46Calvopi~na, K. 16Cantero, J. 18Carr, C. E. 74Case, B. C. 78Chandra, N. 15Chandra, R. 12, 20, 27, 31, 35, 60Chaudhary, C. 27Chazin, W. J. 63Chen, A. A. 74Chen, R. 6Chiranjeevi, P. 34, 40Chiu, T.-P. 81Chu, B. 79Chugh, H. 35Clark, S. 9Coalson, R. D. 45Cojocaru, H. V. 53Colina, R. 18Coutsias, E. 48Dalyan, Y.B. 85Das, S. 2Dantas Machado, A. C. 81DasGupta, D. 17Dasgupta, S. 17Davoodi, M. 36Degtjarik, O. 38, 39Deng, L. 72Dey, S. 4Diskin-Posner, Y. 39Dong, Y. 6Dowson, C. 14Dunker, K. 9Dyudeeva, E. S. 83Fang, R. 5, 6Farkhutdinov, R. G. 43Fawzi, N. L. 10Fayez Aziz, M. 46Fedyaev, V. V. 43Feig, M. 51Fierz, B. 55Finley, D. 6Fogg, J. M. 5Frank, J. 1Freudenreich, C. H. 62

Friesner, R. A. 20Gamazo, P. A. 18Garipova, M. I. 43Ge, P. 2Gevorgyan, E. S. 21, 22, 56, 71Gevorgyan, E. 52, 57Ghosh, I. 28Gnanakaran, S. 47Golfetto, O. 44Golovenko, D. 37, 38, 39Golyshev, V. M. 83Gonen, T. 3Gordan, R. M. 80Grintsevich, E. E. 2Gross, M. L. 8Gruebele, M. 51Gu, C. 45Gupta, M. K. 42Guttula, P. K. 42Hakobyan, N. R. 21Hakobyan, N. 22Haran, T. E. 37, 38Haroutiunian, S.G 85Harvey, S. C. 8Hema, K. 32, 42Hengartner, N. 47H€ogberg, B. 12Hinchliffe, P. 16Hingorani, M. M. 78Hirvonen, V. H.A. 16Hon, J. 6Hong Zhou, Z. 2Honig, B. 44Hosseini, E. 36Hovhannisyan, A. G. 21, 22Ignatov, M. 48Issar, U. 29, 33Iyer, M. S. 67Jane"cek, M. 48Jani, V. 73Jara, K. 9Jarosz, D. F. 10Jayaraj, A. 17Jayaram, B. 17Johari, S. 13Joshi, A. G. 67Joshi, R. 69, 73Jovanovic-Talisman, T. 44

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JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICShttps://doi.org/10.1080/07391102.2019.1604468

Jure"cka, P. 49, 50Kadirvelraj, R. 8Kakkar, R. 29, 33Kamzolova, S. V. 67, 82, 87, 88Kang, H. 6Karapetian, A. T. 75Karapetyan, A. A. 71Karapetyan, A. T. 80Karapetyan, N. H. 86Kasahara, K. 51Kaushal, S. 62Kaushik, A. C. 8Kaushik, R. 17Ke, Y. 11Keul, N. D. 8Kirschner, M. W. 6Klimov, D. K. 25Kompanichenko, V. N. 69Kornoushenko, Y. V. 22, 23Kouzminova, E. A. 61Kozakov, D. 48Krepl, M. 50K€uhrov!a, P. 48, 50Kumar, K. S. 29, 34, 40, 42Kumar, N. 20, 31Kupryushkin, M. S. 76, 83Kuzminov, A. 61Langley, D. R. 6Lennon, K. M. 44Li, Y. 72Li, Z. 55Lim, R. Y. H. 46Liu, S.-Q. 71, 72Lloyd, A. 14Lomzov, A. A. 76, 83L!opez, C. A 47Lu, C. 26Lu, Y. 5, 6Luan, B. 6Lunina, J. N. 82Ma, W. 6MacArdle, S. G. 63Madendran, R. 33Madhukar, G. 28Madhulitha, N. R. 29, 34, 40Mamasakhlisov, Y. 77Mansbach, R. 47Mao, Y. 6Marky, L. A. 73, 74Matinyan, K. S. 56, 71Matinyan, K. 52, 57Maxwell, A. 14McDonald, W. E. 8Mikaelyan, M. S. 87Minasyants, M. V. 75Mirney, L. 59Mitchell, J. 48Mittal, A. 59

Ml!ynsk!y, V. 50Mondal, A. 75Morgunov, G. I. 88Morgunov, I. G. 67, 82, 87, 88Morozov, V. 77Mothay, D. 70Mothay, K.V. D. 18Mu, Y. 78Mughal, F. 46Mukhaelyan, Z. H. 68Mukherjee, S. 17Mukhopadhyay, B.P. 17Mulholland, A. J. 16Munikumar, M. 42Munsamy, G. 37Myers, C. A. 74Naik, V. U. 40Narayanan, S. 18Nawrocki, G. 51Nerkararyan, A. V. 87Newman, H. 14Nikolaev, G. I. 22, 23Norouzi, D. 57Olotu, F. A. 37Oruganty, K. 8Oshima, H. 51Otyepka, M. 48, 49, 50Ouyang, Q. 6Oztug Durer, Z. A. 2Pakala, S. 29Paliwal, S. 64Panchenko, A. 53Panneerselvan, N. 33Parsadanyan, M. A. 75, 77, 79, 84Pasala, C. 29Paukstelis, P. J. 79Petrov, V. V. 65Phillips, R. S. 8Pielak, G. J. 52Poghosyan, G. H. 68, 87Pollack, L. 54Powers, E. T. 51Pradeep, N. 32, 42Pyshnyi, D. V. 76, 83Rahim, F. 30, 36Ramesh 18Ramesh, K. V. 70Rangadurai, A. 80Rangunathan, M. 33Rao, S. 81Re, S. 51Reardon, P. 9Reiling-Steffensmeier, C. 73Reisler, E. 2Rohs, R. 81Rosenberg, S. M. 15Rozenberg, H. 38, 39Ruggerone, P. 47

Safieh, J. 37Sahakyan, V. G. 77Salay, L. E. 63Salimraj, R. 16Schaper Bergman, E. T. 8Schiffer, C. 25Seeman, N. C. 12Sengupta, J. 4Serindag, K. 6Shahinyan, M. A. 77, 80Shahnazaryan, N. H. 84Shakked, Z. 38, 39Shalev-Benami, M. 2Shandilya, A. 17Sharma, N. 69Shekhar, S. 17Shekhar, V. 17Shernyukov, A. V. 76Shevelev, G. Y. 76Shi, H. 80Shigapova, A. I. 43Shirbandi, K. 36Singh, A. 12, 17Singh, S. 60Smith, A. K. 25Soliman, M. E. 37Sonavane, U. B. 69Sonavane, U. 73Soni, A. 17Sood, D. 27Sotnikova, J. M. 43Sowdhamini, R. 67Spencer, J. 16"Sponer, J. 48, 49, 50Srinivasan, K. 4Stepanova, N. N. 82, 87, 88Su, X. A. 62Subbarao, N. 28Subramaniam, S. 1Sugita, Y. 51Sun, R. 55Swargam, S. 32, 42Tao, Y. 71, 72Thayer, K. M. 6Timp, W. 59Tomar, V. 31Tomashevsky, A. A. 65Tonoyan, Sh. 77Tooke, C. L. 16Torosyan, A. L. 86Torosyan, M. A. 80Tort, F. L. 18Tuzikov, A. V. 22Umamaheswari, A. 29, 32, 34, 40, 42Usanov, S. A. 23van der Kamp, M. W. 16Vardevanyan, P. O. 77, 79, 84Vashishtha, A. 13


Veisi, A. 30Verkhivker, G. M. 24Victoria, M. 18Wakefield, D. L. 44Wei, D. Q. 8Wilson, B. S 47Wood, Z. A. 8Wu, J. 6Xia, Y.-L. 71Xu, S. 26Xu, Z. 26Yakupova, A. B. 43Yakushevich, L. V. 81

Yan, H. 11Yavroyan, Z. V. 21Yavroyan, Z. 22Ye, R. 62Yu, I. 51Zakiryanov, F. K. 81Zarezade, V. 30Zechiedrich, L. 5Zgarbov!a, M. 49, 50Zgurskaya, H. I. 16, 47Zhang, X.-Y. 71Zhang, Z. 26Zhao, H. 61

Zheng, L. 78Zheng, T. 45Zhong, A. 63Zhou, X. 26Zhu, Y. 6Zhuang, X. 58Zhurkin, V. B. 57Zidovska, A. 58Zilman, A. 45Zou, L. 62Zunini, M. P. 18


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