Download - David Cooper University of Virginia
![Page 1: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/1.jpg)
The Integrated Center for Structure and Function Innovation:A PSI-2 Specialized Technology Center
David Cooper
University of Virginia
![Page 2: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/2.jpg)
ISFI Mission StatementThe Integrated Center for Structure and Function Innovation (ISFI) is an NIH Protein Structure Initiative Specialized Center focused on developing and applying a set of synergistic technologies organized to overcome recognized bottlenecks in structure determination at the key steps of production of soluble protein and protein crystallization.
Addressing high-throughput bottlenecks:Protein Solubility and Crystallization
• Only 50.5% of “Expressed” proteins are soluble
• Only 35.5% of “Purified” proteins produce crystals
• Only 18.8% of “Purified” proteins produce X-ray quality crystals
Target DB Statistics
# o
f P
rote
ins
Data from Target DB as of Nov. 20, 2006
![Page 3: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/3.jpg)
Los Alamos National Lab
University of Chicago
University of Virginia
Lawrence Livermore National Laboratory
University of California
Los Angeles
The ISFI
Lawrence Berkeley National Laboratory
![Page 4: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/4.jpg)
Los Alamos
UChicago
The University of Virginia
LBNL
LLNL
UCLA
The ISFI
The Joint Center for Structural Genomics
Midwest Center for Structural Genomics
![Page 5: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/5.jpg)
ISFI Protein Pipeline
Los AlamosProtein Production
Facility
UCLAAnalysis of protein complexes
Co-expression of partners
UChicagoCrystallization
chaperone design
LANLDirected EvolutionProtein Production
UVAProtein surface
engineering
LBNLData Collection
Facility
LLNLCrystallization
Facility
![Page 6: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/6.jpg)
Los Alamos: Geoff Waldo et alFinding Soluble Domains using a HT pipeline
LANL
![Page 7: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/7.jpg)
![Page 8: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/8.jpg)
Polyketide Synthase Example
![Page 9: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/9.jpg)
UCLA: David Eisenberg et alIdentification and Crystallization of Protein Complexes
•ProLinks 3.0 http://mysql5.mbi.ucla.edu/A database of inferred functional linkages
Uses: Phylogenetic Profiles Rosetta Stone Gene neighbor Gene cluster
•Crystallization of Complexes Cloning partners identified by ProLinksCo-expression using modified Duet vectorsCrystallization of predicted functional complexes
UCLA
![Page 10: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/10.jpg)
Prolinks identifies 44% (107/242) of PDB prokaryotic complexes
Benchmarking ProLinks Identification of Prokaryotic PDB Complexes
PDB polypeptide chains (17,844 unique chains)Source: 8/2004
Identify functionallylinked sequences
(BLAST against Prolinks)
>= 1 High confidencefunctional linkage
Source organism presentin both PDB and Prolinks
Group sequences by PDB structure(24,475 structures)
NoFuture studies
Identification ofFunctionally
Linked Proteins
Filtering
Yes
Structures w/ >= 2 different chains (complexes)
782 complexes
242 non-redundant complexes
107 non-redundant complexes
NoFuture studies
NoFuture studies
Prokaryotic filter:
Prolinks filter
Biological filter
Yes
http://mysql5.mbi.ucla.edu/
![Page 11: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/11.jpg)
Co-crystallization of predicted complexes
![Page 12: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/12.jpg)
University of Chicago: Tony Kossiakoff et al Chaperone-Assisted CrystallographyAntibody fragments are produced from phage display experiments. This
method has several advantages: Fast antibody production. Improvement in crystal formation. Capability of coupling biochemical studies to selection. Acquisition of necessary phase information for structure
determination of complex (via Molecular Replacement or SeMet antibodies)
Target Validation and Prep1 week
Automated Screening w/KingFisher (3 rounds)
Confirm Enrichment of Hits1.5 weeks
Subclone into Expression Vectors1 week
HTP Protein ProductionBIAcore Assay0.5 week
UChicago
![Page 13: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/13.jpg)
High-Affinity S/Y antibodies
Target BC DE FG Kd
(nM) MBP SYSSSVS GSKS YSYYYYYYSS 18 SYSSVY GSKS YSYYYYYYSS 23 SSYYYYYVS GYSS YSYSSYYSYYYS 20 ySUMO YYSYYSYSVS YYYS YSSYSSSSYS 14 SSSSVS SYYS YYYSYYYYYSYS 7 hSUMO4 SYYYVS SYYS YYSSYYSSYYYSYS 22 SYYYVS SYYS YYYYSYYYYYYS 7
A reduced code of amino acids can be used to generate binding sites with high affinities.A binary code works great! (Ser / Tyr)
FABsfor14
MCSGTargets
FN3 affinities for 3 targets
![Page 14: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/14.jpg)
Two strategies for protein crystallization:
UVA
UVA: Zygmunt Derewenda et al Surface Entropy Reduction
Systematically altering the protein surface to facilitate crystallization
Varying the protein parameter• Homologues• Different construct ends• Reductive Methylation • Alanine scanning• Directed Evolution• Rational Mutagenesis
![Page 15: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/15.jpg)
Promotes crystallization by altering surface features that inhibit crystallization.
Large, flexible residues on the surface can inhibit crystallization.
Lysine and Glutamate are primarily responsible for the “entropy shield”
Candidate Proteins: Soluble and purify well Difficult to crystallize or diffract
poorly Contain a cluster of highly-
entropic residues
Surface Entropy ReductionSystematically altering the protein surface to facilitate
crystallization
Lysine GlutamateRotamers Rotamers
![Page 16: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/16.jpg)
Our Model Protein -- RhoGDI
Observations from initial experiments. Mutated residues are often found at or in crystal contacts. Single mutations may change the kinetics of crystallization, but
double and triple mutations lead to new crystal forms.
Meets all SER criteria Rich in lysines (10.1%) and glutamates (7.9%)
(average incidence of 7.2% and 3.7%, respectively) It took years to get a poorly-diffracting wild-type crystal.
(Longenecker, et al Acta Cryst. D57:679-688. 2001)
(Mateja, et al Acta Cryst. D58:1983-91. 2002)
![Page 17: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/17.jpg)
The RGSL domain of PDZRhoGEFLongenecker KL, et al. & Derewenda Z.S. Structure (2001) 9:559-69
The LcrV antigen of the plague-causing bacterium Yersinia pestisDerewenda, U. et al. & Waugh, D.S. Structure (2001) 9:559-69
Product of the YkoF B. subtilis gene Devedjiev, Y. et al. & Derewenda, Z.S. J Mol Biol (2004) 343:395-406
Product of the YdeN B. subtilis gene Janda, I. et al. & Derewenda, Z.S. Acta Cryst (2004) D60: 1101-1107
Product of the Hsp33 B. subtilis gene Janda, I. et al. & Derewenda, Z.S. Structure (2004) 12:1901-1907
The product of the YkuD B. subtilis gene Bielnicki, J. et al. & Derewenda, Z.S. Proteins (2006) 1:144-51
Human Doublecortin N-terminal domainCierpicki, T. et al, & Derewenda, Z.S. Proteins (2006) 1:874-82
The Ohr protein of B. subtilisCooper, D. et al. & Derewenda, Z.S. in preparation
Human NudC C-terminal domainZheng, M. et al. & Derewenda, Z.S. in preparation
APC1446 -- Crystals diffracting to 3.0 Å, but unsolved.
**MCSG Targets**
Novel proteins crystallized by SER:
![Page 18: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/18.jpg)
RGSL domain of PDZ-RhoGEF Structure 9:559-69 (2001)
YkoF JMB 343:395-406 (2004)
LcrV Structure 12:357-8 (2004)
Hsp33 Structure 12:1901-7 (2004)
The recurrence of crystal contacts involving mutated sites validates the hypothesis that crystallization is facilitated by surface entropy reduction.
![Page 19: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/19.jpg)
Ongoing work: Optimization of the screening protocols Evaluation of other amino acids at crystal forming interfaces:
Alanine, Histidine, Serine, Threonine, Tyrosine Use of bioinformatics for prediction of crystallizable mutants
![Page 20: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/20.jpg)
Ongoing work: Optimization of the screening protocols Evaluation of other amino acids at crystal forming interfaces:
Alanine, Histidine, Serine, Threonine, Tyrosine Use of bioinformatics for prediction of crystallizable mutants
A B C D
E F G H I
![Page 21: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/21.jpg)
The Most successful MutantK138Y, K141Y
•34 hits in the traditional screen•35 hits in the salt screen
•Wild Type•No hits in the traditional screen•1 hit in the salt screen
![Page 22: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/22.jpg)
Conclusions:
Alanine, tyrosine and threonine can be effectively used as crystal-contact mediating residues.
The salt screens produced almost 33% more hits – 242 vs. 183.
Performing traditional and alternative reservoir screening greatly increases the chances of getting a hit and greatly increases the number of conditions that give hits.
At certain surface locations some amino acids seem to nucleate crystal contacts better than others. Thus, different amino acids may be tried at each selected site to increase chances of success.
![Page 23: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/23.jpg)
SER Prediction Server(Luki Goldschmidt, UCLA)
http://nihserver.mbi.ucla.edu/SER/
The server is designed to predict mutations that may increase the likelihood of crystallization. It has many user editable parameters, but is designed to be ready out of the box.
![Page 24: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/24.jpg)
http://nihserver.mbi.ucla.edu/SER/
The SERp Summary Page
The Server presents ranked mutation suggestions. It also links to homologous structures, potential interacting partners and conserved blocks. The secondary structure prediction and blast results are also presented.
![Page 25: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/25.jpg)
http://nihserver.mbi.ucla.edu/SER/
Get Pretty Results – then get crystals.
As of Nov. 21, 260 users have submitted 1430 jobs.
HSP33Structure
![Page 26: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/26.jpg)
Publications by other groups using SER Novel proteins (black) or preparations of higher quality crystal forms (green)
The CUE:ubiquitin complex Prag G et al., & Hurley JH, Cell (2003) 113:609-20
Unactivated insulin-like growth factor-1 receptor kinaseMunshi, S. et al. & Kuo, L.C. Acta Cryst (2003) D59:1725-1730
Human choline acetyltransferaseKim, A-R., et al. & Shilton, B. H. Acta Cryst (2005) D61, 1306-1310
Activated factor XI in complex with benzamidineJin, L., et al. & Strickler, J.E. Acta Cryst (2005) D61:1418-1425
Axon guidance protein MICALNadella, M., et al. & Amzel, M.L. PNAS (2005) 102:16830-16835
Functionally intact Hsc70 chaperoneJiang, J., et al. & Sousa, R. Molecular Cell (2005) 20:513-524
L-rhamnulose kinase from E. coliGrueninger D, & Schultz, G.E. J Mol Biol (2006) 359:787-797
T4 vertex gp24 protein Boeshans, K.M., et al. & Ahvazi, B. Protein Expr Purif (2006) 49:235-43
Borrelia burgdorferi outer surface protein AMakabe, K., et al. & Koide, S. Protein Science, (2006) 15:1907-1914
SH2 domain from the SH2-B murine adapter proteinHu, J., & Hubbard, S.R J Mol Biol, (2006) 361:69-79
Mycoplasma arthriditis-derived mitogenGuo, Y., et al., & Li, H. J., Acta Cryst (2006) F62:238-241
![Page 27: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/27.jpg)
2HDX – SH2-B with JAK2pY813
2HDV – Unliganded SH2-B
Principle in ActionE583A,E584A,(W593H)
![Page 28: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/28.jpg)
Extending the MethodMulti-domain proteins.
2CGJ
2CGK
Red and Green denote domains.
E69A, E70A, R73A
![Page 29: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/29.jpg)
Another Multi-domain Example
Several wild-type crystal forms were “not suitable for x-ray diffraction studies”
Made double Lys->Ala mutant K141A and K142A
“Well diffracting crystals of the mutated protein were readily obtained.”
Red and Green denote domains.
![Page 30: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/30.jpg)
Extending the MethodMutating Multiple Clusters
First MutationsK48A, K60A, K83A,K196ADidn’t work
Added E37S, E45S, K46S,K64S, E104S, K107S, K239S, E240S, and K254S
![Page 31: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/31.jpg)
University of VirginiaZygmunt DerewendaDavid CooperTomek BoczekWonChan ChoiUrszula DerewendaKasia GrelewskaNatalya OlekhnovichGosia PinkowskaMichal ZawadzkiMeiying Zheng
Lawrence Livermore National LaboratoryBrent Segelke Dominique ToppaniMarianne KavanaghTimothy Lekin
Lawrence Berkeley National LaboratoryLi-Wei Hung Evan BurseyThiru
RadhakannanJim WellsMinmin Yu
University of ChicagoAnthony Kossiakoff Shohei Koide Magdalena BukowskaVince CancasciSanjib DuttaKaori EsakiJames HornAkiko KoideValya TerechkoSerdar UysalJingdong Ye
Los Alamos National LaboratoryTom Terwilliger Geoffrey WaldoChang Yub KimEmily AlipioCarolyn BellStephanie
CabantousNatalia FriedlandPawel ListwanJin Ho MoonJean-Denis PedelacqTheresa Woodruff
UCLADavid Eisenberg Daniel AndersonSum ChanLuki GoldschmidtCelia GouldingTom HoltonMarkus KaufmannArturo Medrano-
SotoMaxim PashkovTeng Poh KhengMichael StrongPoh Teng
Acknowledgements
![Page 32: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/32.jpg)
![Page 33: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/33.jpg)
A Success from Screening Alone
A MCSG abandoned target.Wild-type crystallized only in the salt screen!
![Page 34: David Cooper University of Virginia](https://reader036.vdocument.in/reader036/viewer/2022062315/56814c5d550346895db97c62/html5/thumbnails/34.jpg)
Meets all SER criteria Rich in lysines 10.1% and glutamates 7.9% average incidence
of 7.2% and 3.7%, respectively It took years to get a poorly diffracting wild-type crystal
Previous Successes w/ RhoGDI