David Cooper University of Virginia

1. The Integrated Center for Structure and Function Innovation:A PSI-2 Specialized Technology Center David Cooper University of Virginia

2. Addressing high-throughput bottlenecks:Protein Solubility and Crystallization Target DB Statistics • 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 # of Proteins Data from Target DB as of Nov. 20, 2006 ISFI Mission Statement The 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.

3. The ISFI Lawrence Berkeley National Laboratory Lawrence Livermore National Laboratory University of Chicago University of Virginia University of California Los Angeles Los Alamos National Lab

4. The ISFI UChicago LBNL LLNL The Joint Center for Structural Genomics Midwest Center for Structural Genomics The University of Virginia UCLA Los Alamos

5. ISFI Protein Pipeline UCLA Analysis of protein complexes Co-expression of partners UChicago Crystallization chaperone design Los Alamos Protein Production Facility LANL Directed Evolution Protein Production UVA Protein surface engineering LLNL Crystallization Facility LBNL Data Collection Facility

6. LANL Los Alamos: Geoff Waldo et alFinding Soluble Domains using a HT pipeline

8. Polyketide Synthase Example

9. UCLA: David Eisenberg et alIdentification and Crystallization of Protein Complexes UCLA • 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 ProLinks • Co-expression using modified Duet vectors • Crystallization of predicted functional complexes

10. Benchmarking ProLinks Identification of Prokaryotic PDB Complexes Prolinks identifies 44% (107/242) of PDB prokaryotic complexes Identification ofFunctionally Linked Proteins PDB polypeptide chains (17,844 unique chains)Source: 8/2004 Identify functionallylinked sequences(BLAST against Prolinks) Group sequences by PDB structure(24,475 structures) Structures w/ >= 2 different chains (complexes) Prokaryotic filter: No Filtering Future studies 782 complexes Yes Source organism presentin both PDB and Prolinks Biological filter No Future studies 242 non-redundant complexes Yes >= 1 High confidencefunctional linkage Prolinks filter No Future studies 107 non-redundant complexes • http://mysql5.mbi.ucla.edu/

11. Co-crystallization of predicted complexes

12. University of Chicago: Tony Kossiakoff et al Chaperone-Assisted Crystallography UChicago Target Validation and Prep 1 week Automated Screening w/ KingFisher (3 rounds) Confirm Enrichment of Hits 1.5 weeks Subclone into Expression Vectors 1 week HTP Protein Production BIAcore Assay 0.5 week Antibody 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)

13. High-Affinity S/Y antibodies A reduced code of amino acids can be used to generate binding sites with high affinities.A binary code works great! (Ser / Tyr) FABs for 14 MCSG Targets FN3 affinities for 3 targets

14. UVA UVA:Zygmunt Derewenda et alSurface Entropy Reduction Systematically altering the protein surface to facilitate crystallization Two strategies for protein crystallization: • Varying the protein parameter • Homologues • Different construct ends • Reductive Methylation • Alanine scanning • Directed Evolution • Rational Mutagenesis

15. Surface Entropy ReductionSystematically altering the protein surface to facilitate crystallization • 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 Lysine Glutamate Rotamers Rotamers

16. The E2A series Our Model Protein -- RhoGDI • 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. • 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. The K2A series (Longenecker, et al Acta Cryst. D57:679-688. 2001) (Mateja, et al Acta Cryst. D58:1983-91. 2002)

17. The RGSL domain of PDZRhoGEF Longenecker KL, et al. & Derewenda Z.S. Structure (2001) 9:559-69 The LcrV antigen of the plague-causing bacterium Yersinia pestis Derewenda, U. et al. & Waugh, D.S. Structure (2001) 9:559-69 Product of the YkoFB. subtilis gene Devedjiev, Y. et al. & Derewenda, Z.S. J Mol Biol (2004) 343:395-406 Product of the YdeNB. subtilis gene Janda, I. et al. & Derewenda, Z.S. Acta Cryst (2004) D60: 1101-1107 Product of the Hsp33B. subtilis gene Janda, I. et al. & Derewenda, Z.S. Structure (2004) 12:1901-1907 The product of the YkuDB. subtilis gene Bielnicki, J. et al. & Derewenda, Z.S. Proteins (2006) 1:144-51 Human Doublecortin N-terminal domain Cierpicki, T. et al, & Derewenda, Z.S. Proteins (2006) 1:874-82 The Ohr protein of B. subtilis Cooper, D. et al. & Derewenda, Z.S. in preparation Human NudC C-terminal domain Zheng, M. et al. & Derewenda, Z.S. in preparation APC1446 -- Crystals diffracting to 3.0 Å, but unsolved. **MCSG Targets** Novel proteins crystallized by SER:

18. The recurrence of crystal contacts involving mutated sites validates the hypothesis that crystallization is facilitated by surface entropy reduction. Hsp33 Structure 12:1901-7 (2004) RGSL domain of PDZ-RhoGEF Structure 9:559-69 (2001) YkoF JMB 343:395-406 (2004) LcrV Structure 12:357-8 (2004)

19. 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

20. 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

21. The Most successful Mutant • K138Y, 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

22. 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.

23. SER Prediction Server(Luki Goldschmidt, UCLA) 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. http://nihserver.mbi.ucla.edu/SER/

24. 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. http://nihserver.mbi.ucla.edu/SER/

25. Get Pretty Results – then get crystals. HSP33 Structure As of Nov. 21,260 users have submitted 1430 jobs. http://nihserver.mbi.ucla.edu/SER/

26. Publications by other groups using SERNovel 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 kinase Munshi, S. et al. & Kuo, L.C. Acta Cryst (2003) D59:1725-1730 Human choline acetyltransferase Kim, A-R., et al. & Shilton, B. H. Acta Cryst (2005) D61, 1306-1310 Activated factor XI in complex with benzamidine Jin, L., et al. & Strickler, J.E. Acta Cryst (2005) D61:1418-1425 Axon guidance protein MICAL Nadella, M., et al. & Amzel, M.L. PNAS (2005) 102:16830-16835 Functionally intact Hsc70 chaperone Jiang, J., et al. & Sousa, R. Molecular Cell (2005) 20:513-524 L-rhamnulose kinase from E. coli Grueninger 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 A Makabe, K., et al. & Koide, S. Protein Science, (2006) 15:1907-1914 SH2 domain from the SH2-B murine adapter protein Hu, J., & Hubbard, S.R J Mol Biol, (2006) 361:69-79 Mycoplasma arthriditis-derived mitogen Guo, Y., et al., & Li, H. J., Acta Cryst (2006) F62:238-241

27. Principle in ActionE583A,E584A,(W593H) 2HDV – Unliganded SH2-B 2HDX – SH2-B with JAK2pY813

28. Extending the MethodMulti-domain proteins. Red and Green denote domains. E69A, E70A, R73A 2CGK 2CGJ

29. 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.

30. Extending the MethodMutating Multiple Clusters • First Mutations K48A, K60A, K83A,K196A Didn’t work • Added E37S, E45S, K46S,K64S, E104S, K107S, K239S, E240S, and K254S

31. Acknowledgements University of Virginia Zygmunt Derewenda David Cooper Tomek Boczek WonChan Choi Urszula Derewenda Kasia Grelewska Natalya Olekhnovich Gosia Pinkowska Michal Zawadzki Meiying Zheng Los Alamos National Laboratory Tom Terwilliger Geoffrey Waldo Chang Yub Kim Emily Alipio Carolyn Bell Stephanie Cabantous Natalia Friedland Pawel Listwan Jin Ho Moon Jean-Denis Pedelacq Theresa Woodruff University of Chicago Anthony Kossiakoff Shohei Koide Magdalena Bukowska Vince Cancasci Sanjib Dutta Kaori Esaki James Horn Akiko Koide Valya Terechko Serdar Uysal Jingdong Ye UCLA David Eisenberg Daniel Anderson Sum Chan Luki Goldschmidt Celia Goulding Tom Holton Markus Kaufmann Arturo Medrano-Soto Maxim Pashkov Teng Poh Kheng Michael Strong Poh Teng Lawrence Berkeley National Laboratory Li-Wei Hung Evan Bursey Thiru Radhakannan Jim Wells Minmin Yu Lawrence Livermore National Laboratory Brent Segelke Dominique Toppani Marianne Kavanagh Timothy Lekin

33. A Success from Screening Alone A MCSG abandoned target. Wild-type crystallized only in the salt screen!

34. The K2A series The E2A series Previous Successes w/ RhoGDI • 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