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Crystallization By Surface Engineering Methodology And Application

Crystallization By Surface Engineering Methodology And Application. David Cooper Zygmunt Derewenda Laboratory. Surface Entropy Reduction. G cryst = H cryst – T (S protein + S solvent ) cryst. Aims to promote crystallization by altering surface features that inhibit crystallization

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Crystallization By Surface Engineering Methodology And Application

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  1. Crystallization By Surface EngineeringMethodology And Application David Cooper Zygmunt Derewenda Laboratory

  2. Surface Entropy Reduction Gcryst = Hcryst – T (Sprotein + Ssolvent)cryst • Aims to promote crystallization by altering surface features that inhibit crystallization • Lysines and glutamates 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

  3. 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

  4. Other Successes in Our Lab • The RGSL domain of PDZRhoGEF Longenecker KL, et al. & Derewenda ZS. 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**

  5. Crystallization often involves mutation clusters The recurrence of crystal contacts involving the mutations seems to validate the hypothesis that crystallization is facilitated by surface entropy reduction. RGSL domain Of PDZ-RhoGEF Hsp33 Ykof LcrV

  6. Publications by other groups reporting crystallization of novel proteins (green), or preparations of higher quality crystal forms (grey) of proteins previously crystallized, by the surface engineering approach as of Sept 2006 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

  7. 3 Mutations: E583A, E584A, W593H • 2 Structures • 2HDV -- SH2 domain of SH2-B • 2.0 Å resolution with 2 copies in the ASU • 2HDX – SH2 domain of SH2-B with Jak2 Phosphopeptide • 2.3 Å resolution with 6 copies in the ASU • “Crystals of wild-type SH2-BSH2, either unliganded or liganded, were not obtained.”

  8. 2HDV – Unliganded SH2-B E563A, E564A, W573H

  9. 2HDX – SH2-B with JAK2pY813

  10. Purpose of this study • Evaluate effect of various target residues Alanine, Histidine, Serine, Threonine, Tyrosine • Learn how to select sites • Evaluate Alternative Reservoir Screening

  11. Our Screening Process • Protein concentration ~ 15 mg/ml • Standard Screen • Drops of Super Screen reagent + protein Our Super Screen is very similar to JCSG+ • Reservoir is 100 l of Super Screen reagent • “Salt” Screen • Drops of Super Screen reagent + protein • Reservoir is 100 l of 1.5 M NaCl • The Wild-Type RhoGDI • Failed to crystallize in the Standard Screen • 1 hit in the Salt screen

  12. Discontinued: Solubility Problems The Mutation Clusters A B C D E F G H I

  13. Ala His Ser Thr Tyr Totals

  14. CT Mutations commonly form crystal contacts DY AS FA FH

  15. Other Target Residues? • Phospholipase A2 • Not intentionally a SER structure “Biochemical studies of bovine pancreatic PLA2 (Rogers et al., 1998; Yu et al., 2000) suggested that the lysine-to-methionine substitution of the residues 53, 56, 120 and 121 eliminates the anionic interface preference of the wild type.” • K53M, K56M, K120M, and K121M • Improvement of resolution (1.5Å to 1.1 Å) • Suggests that SER could also be used to introduce methionines for phasing purposes. K Sekar, et al & M.-D Tsai. Acta Cryst D62:717

  16. Subtle effects of the mutations This trimer of dimers occurs with as little as 2 and as many as 6 monomers in the ASU EA1 R32 a=b=129.6 c=166.6 EA2 C2 a=132.2 b=131.4 c=92.5 This packing is seen with one or two monomers in the ASU. Notice the same dimer. FH P3221 a=b=75.2 c=91.6 CY P3121 a=b=77.3 c=171.7

  17. Observations from this study • Alanines, Tyrosine and Threonines are good choices for the target residue. • Performing traditional and alternative reservoir screening greatly increases the chances of getting a hit and greatly increases the number of conditions that give hits. • The as yet unexplainable preference for a particular target residue for some mutation clusters suggests that multiple target residues should be tried for each cluster. • Recommendation: Before pursuing mutations, try alternative reservoir screening. If that fails, use the server to pick a mutation site and use 2 or more target residues.

  18. A Success from Screening Alone A MCSG abandoned target. Wild-type crystallized only in the salt screen! Solve / Resolve_build did the trick!

  19. Few Sites are Predicted / Needed OhrB Hsp33

  20. Few Sites are Predicted / Needed Yden YkoF Because many proteins have very few potential sites, trying several target residues for one cluster may be the only option.

  21. IA B D/C EF G H There are some exceptions . . . Like RhoGDI. In this case, you could either use either multiple target residues, multiple clusters or a combination.

  22. Current work:Server Validation • Test Top Hits for RhoGDI and Others • K98,K99,Q100 (no longer the top hit) • Threonine – 6 standard – 4 salt – 7 unique • Tyrosine – No hits. Need to test current tops hits. • New Targets: • Selected MCSG targets that could be purified, but failed to crystallize. • Eliminated proteins with close homologues solved • Submitted sequences to the server and requested those with the highest hits.

  23. Extending the MethodMulti-domain proteins. Red and Green denote domains. 2CGK 2CGJ

  24. Another Multi-domain Example K141A, K142A For multi-domain proteins of unknown structure, using multiple sites (instead of multiple target residues) may help avoid disrupting domain interfaces.

  25. 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 • Too many alanine mutations can decrease solubility, so using a hydrophilic target residue is wise. Yeah, ISFI 

  26. Acknowledgements • Tomasz Boczek • Kasia Grelewska • Gosia Pinkowska • Gosia Sikorska • Michal Zawadzki • Luki Goldschmidt • David Eisenberg • Zygmunt Derewenda

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