Zurich Center for Structural Biology

February 6–7, 2017

Lecture Hall KOL G 201, Aula University of Zurich Rämistr. 71, 8006 Zurich

12th Symposium Trends in Structural Biology

Dear Participant,

Welcome to the 12th Symposium on “Trends in Structural Biology”. Visualizing structures of cellular components is of key importance to understand cellular function. Recent advances in instrumentation and methods for investigating macromolecular structure and function have revolutionized not only structural biology but also cellular biology and the molecular study of disease mechanisms.

This Symposium is organized by the Zurich Center for Structural Biology that has been established in 2013 following a 12-year program, NCCR Structural Biology (2001 to 2013), which was supported by the Swiss National Science Foundation, ETH Zurich and the University of Zurich. The Zurich Center for Structural Biology is represented by sixteen research groups at the University of Zurich and ETH Zurich and two associated technology platforms that provide high throughput binder selection and crystallization services.

This conference will allow the attendees to gain insights into the cutting edge research aimed at understanding the chemical basis of biological processes. The speakers will also present a broad range of biochemical and biophysical methods used for sample preparation, data acquisition and analysis, and computational modeling. Furthermore, methods for integration of data obtained by a range of techniques (hybrid approaches) that offer new avenues for exploring cellular life and the molecular basis of diseases will be discussed.

We wish you a stimulating symposium with many new scientific insights and hope that you enjoy your time in Zurich and on location at the University of Zurich and ETH Zurich campuses.

Prof. Dr. Nenad Ban Prof. Dr. Andreas Plückthun

12th Symposium Trends in Structural Biology

Monday February 6, 20178:15 Registration

8:45 Andreas Plückthun, UZH Nenad Ban, ETHZ «Welcome Address»

Session 1Chair: Nenad Ban, ETHZ

9:00 Stephen Cusack, EMBL, Grenoble «Structural Insights into RNA Synthesis by the Influenza Virus Transcription-Replication Machine»

9:45 Discussion

10:00 Karl-Peter Hopfner, Gene Center, University of Munich «Structural Mechanisms of Swi2/Snf2 Protein:DNA Remodeller»

10:45 Discussion

11:00 Coffee Break

Session 2Chair: Nenad Ban, ETHZ

11:30 James Williamson, Scripps Research Institute, La Jolla «Parallel Assembly Channels in the Bacterial 50S Ribosomal Subunit»

12:15 Discussion

12:30 Philipp Bieri, ETHZ «The Complete Structure of Chloroplast 70S Ribosome in Complex with Translation Factor pY»

12:45 Lunch Break

Session 3Chair: Ohad Medalia, UZH

14:00 Sriram Subramaniam, NIH, National Cancer Institute, Bethesda

«Recent Advances in Cryo-EM: Applications to Drug Discovery»

14:45 Discussion

15:00 Yagmur Turgay, UZH «The Structure of Lamins in Somatic Cells»

15:15 Coffee Break

Session 4 Chair: Kaspar Locher, ETHZ

16:00 Judy Hirst, MRC Cambridge, UK

«Cryo-EM Structure of Mammalian Respiratory Complex I, an Asymmetric 1 MDa Energy-converting Enzyme»

16:45 Discussion

17:00 Daan Swarts, UZH «Structural Basis for Guide RNA Processing and Target DNA Binding by Cpf1»

17:15 Sébastien Campagne, ETHZ «Structural Studies of Alternative Splicing Regulation»

17:30 Roderick Lim, Bio Center Basel «Spatiotemporal Dynamics of the Nuclear Pore Complex Transport Barrier»

17:30 End of Day 1

12th Symposium Trends in Structural Biology

Tuesday February 7, 2017Session 5Chair: Andreas Plückthun, UZH

9:00 Jesse Rinehart, Yale School of Medicine, West Haven «A General Synthetic Gateway to Study the Function of Protein Phosphorylation»

9:45 Discussion

10:00 Dorothee Kern, Brandeis University, Waltham «Evolution of Kinase Energy Landscapes over Several Billion Years»

10:45 Discussion

11:00 Coffee Break

Session 6Chair: Andreas Plückthun, UZH

11:30 Todd Yeates, UCLA, Los Angeles «Giant Protein Assemblies in Nature and by Design»

12:15 Discussion

12:30 Yufan Wu, UZH «Adapting DARPin-based Rigid Fusions for Crystallography»

12:45 Lunch Break

Session 7Chair: Raimund Dutzler, UZH

14:00 Stephen Long, Sloane Kettering Cancer Center, NY «Structure and Function of BEST1: a Calcium Activated Chloride Channel»

14:45 Discussion

15:00 Cristina Manatschal, UZH «Structural and Mechanistic Basis of Proton-coupled Metal Ion Transport in the SLC11/NRAMP Family»

15:15 Xavier Deupi, PSI «Molecular Mechanisms of G Protein-coupled Receptor Activation»

15:30 Franziska Zosel, UZH «Dynamics and Interactions of Intrinsically Disordered Proteins Probed with Single-molecule Spectroscopy»

15:45 Nenad Ban, ETHZ «Concluding Remarks»

16:00 End of Meeting

Abstracts of Talks

Structural Insights into RNA Synthesis by the Influenza Virus Transcription-Replication Machine

Stephen Cusack, FRS European Molecular Biology Laboratory, Grenoble The ‘flu is caused by the highly infectious, rapidly evolving and potentially dangerous influenza virus. We study the molecular mechanisms by which the viral RNA-dependent RNA polymerase transcribes and replicates the viral genome. This is not only of fundamental interest but will also help understand avian to human interspecies transmission of the virus and promote development of new anti-influenza drugs. Influenza, which is a member of the Orthomyxoviridae family of segmented negative strand RNA viruses (sNSV), continues to have an enormous impact on world-wide public health. The eight distinct genome segments are individually packaged into ribonucleoprotein particles (RNPs), which are the functional replication units. Transcription, generating capped and poly-adenylated viral mRNAs, and replication, generating full-length genome or antigenome copies (vRNA and cRNA respectively), are performed by the same virally encoded RNA-dependent RNA polymerase. In general, sNSV polymerases have two unique features. Firstly, they perform transcription by the ‘cap-snatching’ mechanism, whereby short 5′ capped RNA fragments are cleaved from host cell mRNA by an endonuclease intrinsic to the polymerase and then used to prime synthesis of viral mRNAs. Secondly, they recognise each genome segment via their highly conserved, quasi-complementary 3′ and 5′ extremities, known as the promoter. Several recent crystal structures of promoter bound influenza A and B polymerases that shed light on multiple aspects of polymerase function will be discussed and their implication for novel anti-viral drug design targeting directly viral replication highlighted. Most recently we have shown that to gain access to nascent capped transcripts for ‘cap-snatching’ in the infected cell nucleus, influenza polymerase directly binds the phosphorylated C-terminal domain of cellular RNA polymerase II. References: 1. Structure of influenza A polymerase bound to the viral RNA promoter. Pflug, A. et al., Nature 2014. 2. Structural insight into cap-snatching and RNA synthesis by influenza polymerase. Reich, S. et al., Nature 2014. 3. Structural insights into bunya virus replication and its regulation by the vRNA promoter. Gerlach, P. et al. Cell 2015. 4. Influenza polymerase can adopt an alternative configuration involving a radical repacking of PB2 domains. Thierry, E. et al., Mol Cell. 2016. 5. Structural analysis of specific metal chelating inhibitor binding to the endonuclease domain of influenza pH1N1 (2009) polymerase. Kowalinski, E. et al., PLoS Pathog. 2012. 6. Structural basis for an essential interaction between influenza polymerase and Pol II C-terminal domain. Lukarska, M. et al., Nature, in press.

Brief Biography: Dr. Stephen Cusack is Head of the Grenoble Outstation of the European Molecular Biology Laboratory (EMBL) and Group Leader in structural biology of protein-RNA complexes and viral proteins. The Cusack group uses X-ray crystallography to study the structural biology of protein-RNA complexes involved in RNA virus replication, innate immunity and cellular RNA metabolism. Stephen Cusack obtained his Ph.D. in 1976 at the Imperial College, London, UK. He then joined the EMBL Grenoble as a Postdoc and was promoted to Group Leader and Head of the Outstation in 1989.

Structural Mechanisms of Swi2/Snf2 Protein:DNA Remodeller

Karl-Peter Hopfner

Gene Center and Department of Biochemistry, University of Munich

Swi2/Snf2 ATPases are molecular machines that remodel substrate DNA:protein complexes. Most prominently, Swi2/Snf2 chromatin remodeller alter the position and composition of nucleosomes on DNA in a genome wide manner and ensure the correct structural organisation and dynamic properties of chromatin. Swi2/Snf2 remodeller form a very large and diverse family, ranging from smaller single subunit enzymes to large multisubunit chromatin remodeller such as the INO80 complex. INO80/SWR1 family chromatin remodellers are complexes composed of >15 subunits and molecular masses exceeding 1 MDa. They have important roles in transcription, replication and DNA repair by exchanging the histone variants H2A and H2A.Z, as well as other emerging activities such as nucleosome positioning and sliding, but their mechanism, specificity and regulation is poorly understood. In addition, due to their size, flexibility and molecular complexity, megadalton chromatin remodeller are a formidable challenge for structural studies. Using two examples, the single subunit remodeller Mot1 and the INO80 complex, I will discuss recent progress and our current understanding of the targeting, regulation and chemo-mechanical mechanism of remodellers with a focus on integrative structural analysis.

Brief Biography: Dr. Karl-Peter Hopfner is Full Professor at the Gene Center and Department of Biochemistry at the University of Munich. The research of his group aims at understanding the molecular and structural mechanisms how the cellular DNA repair and antiviral RNA sensing protein machineries detect, signal and repair or remove malignant nucleic acids such as damaged DNA and viral RNA. To investigate these processes, his lab uses a variety of techniques such as X-ray crystallography, Cryo-EM, SAXS, CX-MS and biophysical/biochemical methods.

Dr. Hopfner obtained his Ph.D. in Biochemistry at the University of Munich in 1997. He joined the Scripps Research Institute in La Jolla to conduct postdoctoral studies before returning to Munich University as a Tenure Track Professor in 2001. Dr. Hopfner was promoted to Associate Professor at the Gene Center in 2005 and to Full Professor in 2007. Since 2015, he is Director of the Gene Center.

Parallel Assembly Channels in the Bacterial 50S Ribosomal Subunit James R. Williamson Department of Integrative, Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA Using deletion strains and genetic perturbations, we are studying the process of ribosome biogenesis in E. coli. By limitation of bL17 expression, and by deletion of the helicase assembly factor SrmB, pre-50S intermediates accumulate due to bottlenecks in assembly. Analysis of these intermediates isolated from sucrose gradient fractions using quantitative mass spectrometry, SHAPE-SEQ, and cryo-electron microscopy has revealed a striking distribution of intermediates. Using this combination of approaches, the protein composition, RNA conformation, and overall structure of the intermediates can be determined. Single Particle Classification of cryo-EM data can deconvolute the heterogeneous collection of intermediates, resulting in the structure of 13 distinct intermediates at < 5Å resolution. Analysis of this set of structures reveals the order of 23S domain folding, including sequential and parallel elements, as well as a structural basis for protein binding dependencies. The active site of the 50S subunit forms last, but it is strongly coupled to protein binding at distal positions in the ribosome. This broadly based biophysical approach offers striking insights into the process of ribosome assembly in bacterial cells. Reference: Davis Joseph H, Tan Yong Z, Carragher B, Potter Clinton S, Lyumkis D, Williamson James R. Modular Assembly of the Bacterial Large Ribosomal Subunit. Cell 167:1610-22.e15

Brief Biography: Dr. Williamson is a Professor in the Departments of Integrative Structural & Computational Biology and Chemistry, and is a member of the Skaggs Institute for Chemical Biology at The Scripps Research Institute in La Jolla, CA. Dr. Williamson received his Ph.D. in Chemistry from Stanford University in 1988, and following postdoctoral work at the University of Colorado, he joined the faculty in the Chemistry Department at the Massachusetts Institute of Technology in 1990, where he attained the rank of Associate Professor with tenure in 1997. Dr. Williamson moved to TSRI in 1998 as

Professor, and in 2001 he became the Associate Dean for the Chemistry Program. In 2008, he became Dean of Graduate and Postdoctoral Studies for the Kellogg School of Science and Technology at TSRI and in 2015 became the Vice President for Academic Affairs. Dr. Williamson’s research involves the study of RNA structure, RNA-protein interactions, and RNA-ligand interactions using biochemistry, biophysics, and structural biology approaches.

The Complete Structure of the Chloroplast 70S Ribosome in Complex with Translation Factor pY Philipp Bieri, Marc Leibundgut, Martin Saurer, Daniel Boehringer, Nenad Ban Institute of Molecular Biology and Biophysics, ETH Zurich Chloroplasts are cellular organelles of plants and algae that are responsible for energy conversion and carbon fixation by the photosynthetic reaction. As a consequence of their endosymbiotic origin, they still contain their own genome and the machinery for protein biosynthesis. Here, we present the atomic structure of the chloroplast 70S ribosome prepared from spinach leaves and resolved by cryo-EM at 3.4 Å resolution. The complete structure reveals the features of the 4.5S rRNA, which probably evolved by the fragmentation of the 23S rRNA, and all five plastid-specific ribosomal proteins (PSRPs). These proteins, required for proper assembly and function of the chloroplast translation machinery, bind and stabilize rRNA including regions that only exist in the chloroplast ribosome. Furthermore, the structure reveals plastid-specific extensions of ribosomal proteins that extensively remodel the mRNA entry and exit site on the small subunit as well as the polypeptide tunnel exit and the putative binding site of the signal recognition particle on the large subunit. The translation factor pY, involved in light and temperature dependent control of protein synthesis, is bound to the mRNA channel of the small subunit and interacts with 16S rRNA nucleotides at the A- and P-site, where it protects the decoding center and inhibits translation by preventing tRNA binding. The small subunit is locked by pY in a non-rotated state, in which the intersubunit bridges to the large subunit are stabilized.

Recent Advances in Cryo-EM: Applications to Drug Discovery Sriram Subramaniam National Cancer Institute, NIH, Bethesda, MD 20892 Recent breakthroughs in the field of cryo-electron microscopy (cryo-EM) provide new prospects for determination of the structures of a variety of macromolecular assemblies and offer unprecedented opportunities for drug discovery. The prospect that the determination of protein structures to atomic resolution will no longer be limited by size, or by the need for crystallization represents a significant and exciting horizon in structural biology. I will discuss the application of these methods to analyse structures of a variety of biologically and medically relevant multi-protein complexes and membrane protein assemblies, which have historically represented the most challenging frontier in structural biology. Selected publications: Merk A., Bartesaghi A, Banerjee S, Falconieri V, Rao P, Davis M, Pragani R, Boxer M, Earl LA, Milne JLS, Subramaniam S (2016) Breaking cryo-EM resolution barriers to facilitate drug discovery. Cell, 165 1698-1707. Matthies D, Dalmas, O, Borgnia, MJ, Dominik, PK, Merk, A, Rao, P, Reddy, BG, Islam, S., Bartesaghi, A, Perozo, E, Subramaniam, S (2016) Cryo-EM Structures of the Magnesium Channel CorA Reveal Symmetry Break Upon Gating. Cell, 164, 747-756. Banerjee, S, Bartesaghi, A, Merk, A, Rao, P. Bulfer, SL, Yan, Y, Green, N, Mroczkowski, B, Neitz, RJ, Wipf, P, Falconieri, V, Deshaies, RJ, Milne, JLS, Huryn D, Arkin, M, Subramaniam S (2016) 2.3 Å resolution cryo-EM structure of human p97 and mechanism of allosteric inhibition. Science, 351,871-875. Meyerson JR, Chittori S, Merk A, Rao P, Han TH, Serpe, M, Mayer ML, Subramaniam S (2016) Structural basis of kainate subtype glutamate receptor desensitization. Nature 537:567-571. Bartesaghi A, Merk A, Banerjee S, Matthies D, Wu X, Milne JLS, Subramaniam S (2015) 2.2 Å resolution cryo-EM structure of-galactosidase in complex with a cell-permeant inhibitor. Science 348:1147-1151.

Brief Biography: Dr. Sriram Subramaniam received his Ph.D. in Physical Chemistry from Stanford University and completed postdoctoral training in the Departments of Chemistry and Biology at M.I.T. He is chief of the Biophysics Section in the Laboratory of Cell Biology at the Center for Cancer Research, National Cancer Institute. He holds a visiting faculty appointment at the Johns Hopkins University School of Medicine. His current work is focused on the development of advanced technologies for imaging macromolecular assemblies using 3D electron microscopy, and their application to address fundamental problems in HIV/AIDS and cancer research.

The Structure of Lamins in Somatic Cells Yagmur Turgay Department of Biochemistry, University Zurich The nuclear lamina is a fundamental constituent of metazoan nuclei. It is composed mainly of lamins, which are intermediate filament proteins that assemble into a filamentous meshwork, bridging the nuclear envelope and chromatin. Besides providing structural stability to the nucleus, the lamina is involved in many nuclear activities, including chromatin organization, transcription and replication. However,

the structural organization of the nuclear lamina is poorly understood. Here, we use cryo-electron tomography (cryo-ET) to obtain a detailed view of the organization of the lamin meshwork within the lamina. Data analysis of individual lamin filaments resolves a globular-decorated fiber appearance and shows that A- and B-type lamins assemble into tetrameric 3.5 nm thick filaments. Thus, lamins exhibit a structure that is remarkably different from the other canonical cytoskeletal elements. Our findings define the architecture of the nuclear lamin meshwork at molecular resolution, providing insights into their role in scaffolding the nuclear lamina.

CryoEM Structure of Mammalian Respiratory Complex I, an Asymmetric 1 MDa Energy-converting Enzyme Judy Hirst MRC Mitochondrial Biology Unit, Cambridge

Complex I (NADH:ubiquinone oxidoreductase) is one of the largest membrane-bound enzymes in the mammalian cell. It powers ATP synthesis by capturing the free energy produced by electron transfer from NADH to ubiquinone to drive protons across the mitochondrial inner membrane. Mammalian complex I contains 45 subunits. 14 of them are core subunits that house the catalytic machinery and are conserved from bacteria to humans whereas the cohort of 31 supernumerary subunits is specific to mammalian species. In this talk I will describe the structure of mammalian complex I (from Bos taurus) determined by single-particle electron cryomicroscopy, including how all 45 subunits were located and modelled in the density map. Furthermore, computational sorting of the particles led to the identification of different structural classes that are related by subtle domain movements. The different classes reveal conformationally-dynamic regions of the structure that are relevant to catalysis, and correspond to biochemical descriptions of the ‘active-to-deactive’ enzyme transition that occurs during hypoxia and ischaemia. Thus the new structural data provides a foundation for understanding complex I assembly and the effects of mutations that cause clinically-relevant complex I dysfunctions, as well as insights into the roles of the supernumerary subunits and new information on the mechanism and regulation of catalysis.

Brief Biography: Dr. Judy Hirst is Deputy Director of the Medical Research Council’s Mitochondrial Biology Unit in Cambridge (UK). She heads a research group focussing on understanding the structure and molecular mechanism of complex I and its roles in human disease. Dr. Judy Hirst obtained her D. Phil. in Chemistry at Oxford University (UK). She then became Wellcome Trust Research Fellow at The Scripps Research Institute in La Jolla (California) before joining the MRC in 1999. Since 2011 she is also a Fellow and Director of Studies in Chemistry at the Corpus Christi College, Cambridge (UK).

Structural Basis for Guide RNA Processing and Target DNA Binding by Cpf1

Daan Swarts

Department of Biochemistry, University Zurich The CRISPR-associated protein Cas9 has emerged as a powerful genome editing tool. Cas9 is an RNA-guided DNA endonuclease that can be programmed using short RNA molecules to generate double-strand breaks in the genomic DNA of eukaryotic cells. Repair of DNA breaks by non-homologous end joining of homology-directed DNA repair can be exploited to introduce genetic modifications in targeted DNA. More recently, the genome editing potential of Cpf1, another CRISPR-associated protein, has been exploited [1]. In contrast to Cas9, Cpf1 is processes its own RNA guide and generates staggered-end DNA breaks. Furthermore, Cas9 requires a GG-containing motif next to the targeted sequence (Protospacer adjacent motif, PAM), whereas Cpf1 requires a T-rich PAM. Combined, these features make Cpf1 a useful addition to the genome editing toolkit. Recent Cpf1 structures have revealed the overall architecture of Cpf1 as well as its RNA guide binding and PAM recognition mode [2-4]. However, the catalytic mechanism of RNA guide processing as well as the molecular mechanisms of RNA-guided DNA target binding and cleavage remain elusive. To shed light on these mechanisms, we determined crystal structures of Francisella tularensis subsp. novicida (FnCpf1) in a binary complex with RNA guide, and in ternary complex with RNA guide and full-length DNA target [5]. These structures suggest that RNA guide processing involves a metal-independent catalytic mechanism, and demonstrate the structural basis for seed-dependent DNA targeting. Importantly, the structure of the ternary complex reveals the path of the displaced DNA strand, suggesting a mechanism for guide RNA-dependent R-loop formation and target DNA cleavage. Together, our structural observations and corroborating biochemical experiments advance our understanding of Cpf1 endonucleases and establish a mechanistic foundation for their use in genome engineering applications. References: [1] Zetsche, B., et al. (2015). Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163, Issue 3, pp. 759-771 [2] Dong, D., et al. (2016). The crystal structure of Cpf1 in complex with CRISPR RNA. Nature 532, pp. 522-526 [3] Yamano et al., (2016). Crystal structure of Cpf1 in complex with guide RNA and target DNA. Cell 165, Issue 4, pp. 949-962 [4] Gao et al., (2016). Type V CRISPR-Cas Cpf1 endonuclease employs a unique mechanism for crRNA-mediated target DNA recognition. Cell Research 26, pp. 901-913 [5] Swarts, D.C., Van der Oost, J., Jinek M. (manuscript submitted). Structural basis for guide RNA processing and seed sequence-dependent target DNA binding by Cpf1.

Structural Studies of Alternative Splicing Regulation Sébastien Campagne, Emil Dedic, Alain Scaiola, Antoine Clery and Frédéric H-T. Allain Institute of Molecular Biology and Biophysics, ETH Zurich Genes encoding for proteins are transcribed by the RNA polymerase II into pre-messenger RNA that are processed (capped, spliced, cleaved and poyadenylated) to generate the mature mRNA, competent for export in the cytoplasm and translation. Removal of intronic sequences (or splicing) is orchestrated by a dynamic molecular machine called the spliceosome that assembles and disassembles at each splicing event. The recognition of the 5’-splice site by the U1snRNP particle is the first event of spliceosomal assembly, and it is regulated by a plethora of splicing factors that bind the surrounding of specific splicing sites and modulate the splicing reaction. From this regulation originates alternative splicing, a fundamental mechanism that generates the protein wealth of metazoans. Understanding the molecular mechanisms that drive splicing regulation remains a major challenge for structural biology. In order to get structural insights into splicing regulation, we developed a method to reconstitute the complete U1snRNP and to observe it using solution NMR. Although two low resolution crystal structures of U1snRNP have been previously solved (1,2), they did not provide any information on the flexible parts which account for roughly 1/2 of the total mass of the particle. We started to collect NMR parameters on these flexible parts and in combination with other methods (SANS and electron microscopy), we are trying to generate a complete structural model of U1snRNP. Having access to large amounts of U1snRNP allowed us to test interactions with splicing factors. We found that SRSF1, FUS and PTB directly recognize the RNA component of U1snRNP using one of their RNA recognition motifs (RRM), leaving the other domains available for the recognition of the pre-mRNA. FUS and SRSF1 bind to the stem loop III of U1snRNP which represents a new hub for splicing regulation. We could show that both proteins bind simultaneously U1snRNP and that the binding of FUS stimulates the recruitment of SRSF1. A structural model of the ternary complex formed by SRSF1, FUS and the stem loop III has been determined and is supported by in vivo data. Based on the fact that FUS has been shown to be essential to couple transcription and splicing (3), we propose that FUS will act as a RNA chaperone to recruit SRSF1 co-transcriptionally which will stimulate spliceosomal assembly. In case of weak 5’-splice sites, the presence of a GGU or GGA motif at the surroundings could be recognized respectively by the zinc finger of FUS or the pseudo-RRM of SRSF1. Altogether, by providing additional contacts with the pre-mRNA, the presence of splicing factors bound to U1snRNP favor the binding of U1snRNP on non-optimal sites and allow splicing to occur. References: (1) Pommeranz-Krummel et al., Nature (2009) (2) Weber et al., EMBO J. (2010) (3) Yu et al., PNAS (2015)

Spatiotemporal Dynamics of the Nuclear Pore Complex Transport Barrier Yusuke Sakiyama1, Adam Mazur2, Larisa E. Kapinos1 and Roderick Y.H. Lim1 1Biozentrum and the Swiss Nanoscience Institute, University of Basel, Switzerland 2Research IT, Biozentrum, University of Basel, Switzerland Nuclear pore complexes (NPCs) are conduits that regulate the traffic of mRNA, pre-ribosomal proteins and other specific macromolecules between the cytoplasm and nucleus in the eukaryotic cell. Within the pore interior lie numerous intrinsically disordered, barrier-forming proteins known as phenylalanine-glycine nucleoporins (FG Nups). It is unclear how NPCs facilitate selective traffic whilst obstructing non-specific macromolecules because the FG Nups have not been structurally resolved inside the pore. Here, we directly visualize the structural dynamics of FG Nups with single molecule resolution inside native Xenopus laevis oocyte NPCs using high-speed atomic force microscopy (HS-AFM). Importantly, HS-AFM acquires molecular movies at 10 frames per second, which approaches the spatiotemporal resolution of coarse-grained dynamics simulations. Individual NPCs feature eight nuclear filaments that assemble into a nuclear basket at the inner nuclear membrane whereas eight cytoplasmic filaments surround a central channel on its cytoplasmic face. This channel is circumscribed by highly flexible, dynamically fluctuating FG Nups that rapidly elongate and retract, being consistent with the diffusive motion of tethered polypeptide chains. Interestingly, the FG Nups exhibit short-lived entanglements but do not cohere into a cross-linked meshwork. Furthermore, the FG Nups interact with cargo complexes in a highly stochastic manner rather than coalescing around it. Our results suggest that the FG Nup barrier “morphology” effectively depends on the observational frame of reference of each respective cargo (i.e., diffusion rate).

General Synthetic Gateway to Study the Function of Protein Phosphorylation

Jesse Rinehart Yale University School of Medicine, New Haven, CT

Protein phosphorylation encompasses a central cellular language that determines every facet of normal cellular biology and often dictates disease phenotypes. New technologies are bringing researchers closer to understanding how protein phosphorylation functions in normal systems and are defining new paradigms for research and discovery across human disease. We have recently created a technology that enables site-specific incorporation of phosphoserine into proteins by expanding the genetic code of Escherichia coli. We are now applying this technology to understand the properties of phosphoserine across broad classes of human proteins. Our current research aims have benefitted tremendously from our recent improvements to our technology. Advances on all fronts will be discussed with attention to the broad application of recombinant phosphoprotein production for biochemical structure/functional studies of protein phosphorylation.

Selected publications (http://rinehart.yale.edu/publications/): Pirman NL, et al. (2015) A flexible codon in genomically recoded Escherichia coli permits programmable protein phosphorylation. Nature Communications. 6, 8130. Amiram M, et al. (2015) Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids. Nature Biotechnology. 33, 1272–1279. Park HS, et al. (2011) Expanding the genetic code of Escherichia coli with phosphoserine. Science 333:1151-1154.

Brief Biography: Dr. Jesse Rinehart is an Associate Professor in the Department of Cellular & Molecular Physiology at the Yale University School of Medicine with a joint appointment in the Systems Biology Institute. Dr. Rinehart’s research aims to understand and “decode” the roles of protein phosphorylation in humans. His laboratory uses an innovative combination of quantitative phosphoproteomics and synthetic biology study protein phosphorylation in single proteins and protein networks. Recently, research in Dr. Rinehart’s laboratory has been accelerated by their Escherichia coli based technology that enables site-specific incorporation of phosphoserine into human proteins. Dr. Rinehart received his PhD in Molecular Biophysics and Biochemistry from Yale in 2005. He studied protein synthesis and the evolution of the genetic code during his graduate work. He did his postdoctoral research with Richard Lifton in the Department of Genetics at Yale and focused on protein phosphorylation in physiological systems.

Evolution of Kinase Energy Landscapes over Several Billion Years

Dorothee Kern

HHMI/ Brandeis University, Dept. of Biochemistry, Waltham

Allosteric regulation, the process by which a protein’s activity can be modulated by binding of an effector molecule distal to the active site, is vital for cellular signaling. However, its evolution is largely unexplored territory. I will describe our experimental exploration of the evolution over 1.5 billion years of two allosteric regulation mechanisms widely found in the modern protein kinase superfamily, phosphorylation of the activation loop and binding of a regulatory partner protein. Using Ancestral Sequence Reconstruction (ASR) we unravel the origins of allosteric activation including surprising mechanistic features. Moreover, ASR enabled identification of the underlying allosteric network that spans the kinase from the N-terminal to the C-terminal lobes. In the second part of the talk I describe how we exploit this new knowledge for the development of allosteric inhibitors and activators. This latter approach delivered novel kinase inhibitors and activators with extreme specificity and high affinity thereby opening the road to new cancer treatment. Third, I will address the evolution of enzyme catalysis in response to one of the most fundamental evolutionary drivers, temperature. Using ASR, we answer the question of how enzymes coped with an inherent drop in catalytic speed caused as the earth cooled down over 3.5 billion years. Tracing the evolution of enzyme activity and stability from the hot-start towards modern hyperthermophilic, mesophilic and psychrophilic organisms illustrates active pressure versus passive drift in evolution on a molecular level.

Brief Biography: Dr. Dorothee Kern is Professor of Biochemistry at Brandeis University and an Investigator of the Howard Hughes Medical Institute. She received her PhD at the Martin Luther University in Halle, Germany and then carried out her postdoctoral studies at UC Berkeley. She joined the faculty at Brandeis University in 1999.Her research group studies the dynamical nature of proteins with the goal to reveal the interplay between structure, dynamics and function. She has been a major contributor in the experimental characterization of protein dynamics during enzyme catalysis and signaling. Her group is exploring the evolution of complex biological function as well as rational drug design by combining a variety of biophysical methods such as NMR, X-ray, fast kinetics, fluorescence, MD simulation, ancestral reconstruction and life-cell imaging.

Giant Protein Assemblies in Nature and by Design

Todd O. Yeates

UCLA Dept. of Chemistry and Biochemistry, Molecular Biology Institute UCLA-DOE Institute for Genomics and Proteomics, and California NanoSystems Institute Nature has evolved myriad sophisticated structures based on the assembly of protein subunits. Many types of natural protein assemblies have been studied extensively. Viral capsids are one example. A number of equally sophisticated natural protein assemblies are only beginning to be appreciated. Among them is a broad class of giant, capsid-like assemblies referred to as bacterial micro-compartments. They serve as primitive metabolic organelles in many bacteria by encapsulating sequentially acting enzymes within a selectively permeable protein shell. Our laboratory has elucidated key mechanisms of these protein-based bacterial organelles through structural studies. Beyond their biological importance, complex protein assemblies like these have for many years represented an ultimate goal in protein design. By exploiting principles of symmetry that are shared by nearly all natural self-assembling structures, we have developed methods for engineering novel proteins that assemble to form a variety of complex, symmetric architectures. Recent successful designs include hollow protein cages composed of many identical subunits in cubic arrangements. Symmetric materials that extend by growth in two or three dimensions are also possible. Natural protein-based compartments and varied designer containers that can now be engineered offer new prospects for synthetic biology and biomedical applications. Design principles and strategies will be discussed, along with current successes.

Brief Biography: Dr. Yeates earned his PhD in 1988 at UCLA. He then moved to The Scripps Research Institute to do postdoctoral research on the structure of poliovirus. Yeates returned to UCLA in 1990 to join the Faculty in the Department of Chemistry and Biochemistry. His interdisciplinary research has focused on macromolecular structure and computational genomics. His varied research findings include: an explanation for why proteins crystallize in certain favored arrangements; the development of new equations for detecting disorder in x-ray diffraction data from protein crystals; the discovery of thermophilic microbes rich in intracellular disulfide bonds; development of comparative genomics methods; development of designed protein cages or ‘nanohedra’; the discovery of novel topological features such as links and slipknots that stabilize thermostable proteins; and elucidation of the structure of the carboxysome shell and the shells of other bacterial microcompartments, which serves as primitive metabolic organelles inside many bacterial cells. Yeates is a member of the Molecular Biology Institute, the California

Nanosystems Institute, the UCLA-DOE Institute of Genomics and Proteomics, and a Fellow of the American Association for the Advancement of Science.

Adapting DARPin-based Rigid Fusions for Crystallography

Yufan Wu

Department of Biochemistry, University Zurich

The major bottleneck in macromolecular X-ray crystallography is the poor packing of protein molecules, frequently resulting in low quality crystals, and the ensuing problems in phase determination, especially with poorly diffracting crystals. DARPins [1] have been widely used as crystallization aides and are well known for their stability, high expression yield, and their ability to crystallize under various conditions. Despite all advantages, DARPins sometimes fail to provide the necessary polar surface to form crystal contacts due to their small size in comparison to the target protein. To extend the range of potential applications of the DARPins as crystallization chaperones, we have developed rigid fusion proteins linked through shared helices, either with another well crystallizing protein (β-lactamase) [2] or, more versatile, with one or more additional DARPins. As these can all be added in a series of different orientations, we have thereby introduced molecular geometry as an additional screening parameter beyond the traditional methods. Thus, a “tool box” of DARPin-based co-crystallization chaperones with a variety of rigid fusion proteins with different geometries was applied to assist the crystallization of other proteins in a simple ‘cut-and-paste’ manner of the desired DARPin fusion. This protein engineering tool box has already helped in obtaining several protein structures. Moreover, this project has shed light on the fundamental question of how to connect proteins rigidly. Rigid DARPin-DARPin fusions also have great potential in other applications, as the constructs can be used to organize supramolecular complexes of different geometries and offer new insights into the geometric constraints and mechanism of receptor activity. References: 1. Plückthun, A. (2015). Designed Ankyrin Repeat Proteins (DARPins): Binding Proteins for Research, Diagnostics, and Therapy. Annu. Rev. Pharmacol. Toxicol. 55, 489-511. 2. Batyuk, A., Wu, Y., Honegger, A., Heberling, M. M., and Plückthun, A. (2016). DARPin-Based Crystallization Chaperones

Structure and Function of BEST1: a Calcium Activated Chloride Channel Stephen B. Long Memorial Sloan Kettering Cancer Center, New York City The calcium-activated chloride channel BEST1 opens its ion conduction pore in response to elevated calcium levels. Its three-dimensional structure and electrophysiological analysis reveal mechanisms for calcium dependent activation and ion selectivity.

Brief Biography: Dr. Long is Member and Professor of the Memorial Sloan Kettering Cancer Center in New York. His research group uses a combination of cryo-electron microscopy, x-ray crystallography, and functional approaches to study the mechanisms of eukaryotic ion channels involved in calcium signaling and membrane-embedded enzymes. Stephen Long obtained his Ph.D. in Biochemistry at Duke University in 2001. He then joined the Rockefeller Institute, New York as a postdoctoral fellow. In 2007, he became Professor at Memorial Sloan Kettering Cancer Center.

Structural and Mechanistic Basis of Proton-coupled Metal Ion Transport in the SLC11/NRAMP Family Cristina Manatschal, Ines A. Ehrnstorfer and Raimund Dutzler Department of Biochemistry, University Zurich Divalent metal ion transporters (DMTs) of the SLC11/NRAMP family transport iron and manganese across cellular membranes. These proteins are highly conserved across all kingdoms of life and thus likely share a common transport mechanism. Our previous crystal structure of Staphylococcus capitis DMT (ScaDMT) has established the structural relationship of the SLC11 family with the amino acid transporter LeuT and it revealed the location of a conserved transition-metal ion binding site in the center of the transporter. In this structure, ScaDMT adopts an inward-facing conformation. Recently, we have determined the crystal structure of Eremococcus coleocola DMT (EcoDMT) in an outward-facing conformation. Together these structures define the endpoints of the transport cycle. Functional assays with proteoliposomes established EcoDMT as secondary active transporter that couples the symport of Mn2+ and protons with a KM in the low micromolar range. Mutants of residues of the metal ion binding site severely affected both, Mn2+ and proton transport thus suggesting that the transport of protons requires conformational changes of the transporter. Inspection of both structures revealed two protonatable residues close to the metal ion binding site that have changed their accessibility to either side of the membrane as potential candidates for proton acceptors. Mutation of one of these residues, a conserved histidine on α-helix 6b, resulted in metal ion transport that appears to be no longer coupled to protons, which implies that this residue likely plays a central role in proton transport. Taken together, our studies have revealed the conformational changes underlying transition-metal ion transport in the SLC11 family by the alternate access mechanism and they provide important insights into the determinants of its coupling to protons. References: 1. Ehrnstorfer, I.A., Geertsma, E.R., Pardon, E., Steyaert, J. & Dutzler, R. Crystal structure of a SLC11 (NRAMP) transporter reveals the basis for transition-metal ion transport. Nat Struct Mol Biol, 2014 2. Ehrnstorfer, I.A., Manatschal, C., Arnold, F.M., Laederach, J. & Dutzler, R. Structural and mechanistic basis of proton-coupled metal ion transport in the SLC11/NRAMP family, Nature Communications, 2017

Molecular Mechanisms of G Protein-coupled Receptor Activation Xavier Deupi Paul Scherrer Institute, Villigen G protein-coupled receptors (GPCRs) are a large family of transmembrane proteins that trigger cellular signaling responses upon binding of extracellular ligands. Thus, GPCRs act as transmission devices between the environment and the cell interior and, due to this key physiological role, they constitute one of the most important pharmaceutical targets. However, despite recent breakthroughs in GPCR crystallography, the structural and mechanistic aspects of GPCR activation by drugs are not yet well understood. At the Laboratory of Biomolecular Research in the Paul Scherrer Institute, we combine imaging (cryo X-ray and electron tomography), structural (X-ray crystallography, electron microscopy, electron diffraction), biophysical (NMR, BRET), and computational (modeling and molecular dynamics simulations) techniques to study the structure and function of GPCRs at different spatial and temporal scales. In this talk, I will show how we have used NMR and sequence/structure analyses to follow ligand-induced activation in the β1-adrenergic receptor (β1AR) (1). We observe that the response to various ligands is heterogeneous in the vicinity of the extracellular binding pocket, but gets transformed into a homogeneous readout at the intracellular side of the receptor. An analysis of the currently available GPCR crystal structures in inactive and active conformations has allowed us to generalize these findings (2). Our analysis allows us to identify crucial connections in the allosteric signal transmission pathway of ligand-induced GPCR activation, and represents a general experimental method to delineate signal transmission networks at high-resolution in GPCRs. References: 1: Isogai S, Deupi X, Opitz C, Heydenreich FM, Tsai CJ, Brueckner F, Schertler GF, Veprintsev DB, Grzesiek S. Backbone NMR reveals allosteric signal transduction networks in the β1-adrenergic receptor. Nature 2016 Feb 11; 530(7589):237-41. 2: Venkatakrishnan AJ, Deupi X, Lebon G, Heydenreich FM, Flock T, Miljus T, Balaji S, Bouvier M, Veprintsev DB, Tate CG, Schertler GF, Babu MM. Diverse activation pathways in class A GPCRs converge near the G-protein-coupling region. Nature 2016 Aug 25; 536(7617):484-7.

Dynamics and Interactions of Intrinsically Disordered Proteins Probed with Single-molecule Spectroscopy Franziska Zosel Department of Biochemistry, University Zurich Many eukaryotic proteins do not adopt folded structures under native conditions. These “intrinsically disordered proteins” (IDPs) are frequently involved in regulatory cellular processes. We use single-molecule Förster resonance energy transfer (FRET) to directly observe the conformational heterogeneity and binding dynamics of the nuclear coactivator binding domain of CBP/p300 (NCDB), a flexible protein domain involved in transcriptional coactivation. The approach enables us to determine association and dissociation rates, the equilibrium constant of binding, as well as the translational diffusion coefficient from a single experiment. We find that NCBD populates two distinguishable conformations, which are both capable of binding its IDP ligand, the activation domain of the steroid receptor coactivator (ACTR). Furthermore, we study the influence of macromolecular crowding (as mimicked by biocompatible polymers) on the binding reaction between NCBD and ACTR. With increasing size and concentration of the crowding agents, we observe a slow-down of dissociation concomitant with an acceleration of association leading to a six-fold affinity enhancement of the protein complex. This effect can be described by depletion interactions induced by the crowders. Our findings provide an explanation for the remarkable binding promiscuity of NCBD and suggest a general mechanism for the favorable effect of crowding on protein-protein interactions.