Binding Specificity of SH2 Domains Revealed by a Combinatorial Peptide Library Thesis Presented in Partial Fulfillment of the Requirements for the Master’s Degree in the Graduate School of The Ohio State University By Andrew Kunys, B.S. Graduate Program in Chemistry The Ohio State University 2013 Thesis Committee: Dehua Pei, Advisor Ross Dalbey ii Copyright by Andrew Kunys 2013 iii Abstract Src homology 2 Domains (SH2 domains) are modular protein binding domains which recognize a phosphotyrosine and the residues adjacent to the phosphotyrosine. Although they are known to bind to specific phosphopeptides motifs, the exact peptide binding motifs for each of the 120 human SH2 domains are not known. These proteins are associated mainly with cellular signaling affecting processes as disparate as apoptosis and proliferation. In this work, high quality specificity data was obtained from 18 different SH2 domains by screening them individually against a One-Bead-One-Peptide library. Interestingly, no two domains have the same specificity, suggesting that subtle differences mark each domain as unique. Further, most of the domains studied recognize multiple different classes of binding peptides, which implies their role in binding may be more complicated that previously realized. The data obtained is unique, in that no other method can produce data of high enough quality to unambiguously establish the subtle specificity patterns of each domain or decipher the data obtained from a domain which can recognize multiple peptide ligand classes. This data will provide insight into the basic biology of cell signaling and should prove to be a valuable resource to researchers studying in this area. ii Acknowledgements I would like to thank, first and foremost, my mother and father for their love and support. Second, I would like to thank my colleagues and friends in Dr. Pei’s research group. I thank Yanyan Zhang and Pauline Tan for their encouragement, friendship and mentorship. I thank Nicholas Selner and Tao Liu for their encouragement and the many helpful discussions we have shared. I thank Tiffany Waller, Xianwen Chen, and Rinrada Luechapanichkul for their friendship and perspective. Finally, I thank Dr. Dehua Pei for his mentorship and support. iii Vita 2008 ……………………….........................................B.S. Chemistry, University of Michigan 2008 to present ...............................................Graduate Research Associate, Department of Chemistry, The Ohio State University 2008-2009........................................................Graduate Teaching Assistant, Department of Chemistry, The Ohio State University 2012-2013.........................................................Graduate Teaching Assistant, Center for Life Science Education, The Ohio State University Publications Kunys, A.R., Lian, W., and Pei, D. 2012. Specificity profiling of protein-binding domains using one-bead-one-compound Peptide libraries. Curr Protoc Chem Biol 4:331-355. Zhang, Y., Wavreille, A.S., Kunys, A.R., and Pei, D. 2009. The SH2 domains of inositol polyphosphate 5-phosphatases SHIP1 and SHIP2 have similar ligand specificity but different binding kinetics. Biochemistry 48:11075-11083. Fields of Study Major Field: Biological Chemistry iv Table of Contents Abstract ………………………………………………………………………………………….….……………………………………...ii Acknowledgements ……………………………………………………………………….………………….………………………iii Vita ……………………………………………………………………….……………………………………….….………………………iv List of Tables …………………………………………………………………………………………………………………………….vii List of Figures ……………………………………………………………………………………………………………………………..x Introduction SH2 domains discovery and function ………………………………………………………....…………………1 SH2 Structure and specificity determining elements ……………………………………………………3 Traditional methods of determining binding specificities .…………………………………….……….5 Determination of SH2 Specificity via One Bead One Compound libraries …..…………….……8 Experimental methods Materials ……………………………………………………….……………….............................................13 Expression of the SH2 Domains ……………………….…………………………………….……………........14 Purification and labeling of the SH2 Domains ………………………….……….………….…….………14 Covalent Labeling of SH2 Domains …………………………………………………….……..…………….....15 Library Synthesis …………………………………………………………………………………..……..................15 Screening SH2 Domains against the pY library ……………………………………..………………….....16 Partial Edman Degradation …………………………………………………………………..……………………..17 v MALDI-TOF analysis …………………………………………………….………………………………….............18 Results Library Used ….…………………………………………………………………………………………………………….19 Kinase SH2 Domains (Itk, Brk, SRMS, Frk, Btk) ………………………………….……………..……….….19 Adapter SH2 Domains (Gads, GRAP) ……………………………………………………………………………37 Shc Family SH2 Domains (Shc1, Shc2, Shc3, Shc4) ………………………………………….……….….44 Vav3 and SHE ………………………………………………………………………………………………………………59 (PLCG1-N, DAPP1, Blnk, SLA2, SH2D3A ……………………………….……………………………………..71 Discussion ……………………………………………………………………………………………………………………….……….94 Bibliography ………………………………………………………………………………………………………………….…………96 vi List of Tables Table 1. SH2 domains Examined in this study ………………………………………………………………………….23 Table 2. Peptide sequences selected from the Itk-SH2 Domain ………………………………………….…..24 Table 3. Peptide sequences selected from the Brk-SH2 Domain ………………………………….…….……25 Table 4. Peptide sequences selected from the SRMS-SH2 Domain ……………………………….…………26 Table 5. Peptide sequences selected from the Frk-SH2 Domain ………………………………….….……….27 Table6. Peptide sequences selected from the Btk-SH2 Domain ………………………………….….….……28 Table 7. Predicted Binding Partners of the Itk-SH2 Domain (Class 1) ………………….…………..….……31 Table 8. Predicted Binding Partners of the Itk-SH2 Domain (Class 2) ………………………………….……32 Table 9. Predicted Binding Partners of the Brk-SH2 Domain ……………………………………..……….……33 Table 10. Predicted Binding Partners of the SRMS-SH2 Domain ……………………………..………….……34 Table 11. Predicted Binding Partners of the Frk-SH2 Domain …………………………………………….……35 Table 12. Predicted Binding Partners of the Btk-SH2 Domain ………………………………………...….……36 Table13. Peptide sequences selected from the Gads-SH2 Domain …………………………….……………39 Table14. Peptide sequences selected from the GRAP-SH2 Domain …………………………………………40 Table 15. Predicted Binding Partners of the Gads-SH2 Domain ………………………………………..….….42 Table 16. Predicted Binding Partners of the GRAP-SH2 Domain ……………………………………..….……43 Table 17. Peptide sequences selected from the Shc1-SH2 Domain ………………………………………….47 Table 18. Peptide sequences selected from the Shc2-SH2 Domain …………………………..……………..48 Table 19. Peptide sequences selected from the Shc3-SH2 Domain ………………………………………….49 Table 20. Peptide sequences selected from the Shc4-SH2 Domain ………………………………….………50 vii Table 21. Predicted Binding Partners of the Shc1-SH2 Domain (Class 1) ………………………………….53 Table 22. Predicted Binding Partners of the Shc1-SH2 Domain (Class 2) ………………………………….54 Table 23. Predicted Binding Partners of the Shc2-SH2 Domain …………….………………………………….55 Table 24. Predicted Binding Partners of the Shc3-SH2 Domain (Class 1) ………………………………….56 Table 25. Predicted Binding Partners of the Shc3-SH2 Domain (Class 2) ………………………………….57 Table 26. Predicted Binding Partners of the Shc4-SH2 Domain ………………………….…………………….58 Table 27. Peptide sequences selected from the Vav3-SH2 Domain ………………….………………………61 Table 28. Peptide sequences selected from the SHE-SH2 Domain …………………………………..….…..62 Table 29. Predicted Binding Partners of the Vav3-SH2 Domain (Class 1) ………………………………….65 Table 30. Predicted Binding Partners of the Vav3-SH2 Domain (Class 2) ………………………………….66 Table 31. Predicted Binding Partners of the Vav3-SH2 Domain (Class 3) ………………………………….67 Table 32. Predicted Binding Partners of the Vav3-SH2 Domain (Class 4) ………………………………….68 Table 33. Predicted Binding Partners of the SHE-SH2 Domain (Class 1) ………….……………………….69 Table 34. Predicted Binding Partners of the SHE-SH2 Domain (Class 2) …………….…………………….70 Table 35. Peptide sequences selected from the PLCG1-N-SH2 Domain ………………….………………..75 Table 36. Peptide sequences selected from the DAPP1-SH2 Domain ………………….…………………..76 Table 37. Peptide sequences selected from the Blnk-SH2 Domain ………………….……………………….77 Table 38. Peptide sequences selected from the SLA2-SH2 Domain ………………….………………………78 Table 39. Peptide sequences selected from the SH2D3A-SH2 Domain ………………….…………………79 Table 40. Predicted Binding Partners of the PLCG1-N-SH2 Domain (Class 1) ……………………..…….83 Table 41. Predicted Binding Partners of the PLCG1-N-SH2 Domain (Class 2) …………..……………….84 Table 42. Predicted Binding Partners of the DAPP1-SH2 Domain (Class 1) ……………………………….85 Table 43. Predicted Binding Partners of the DAPP1-SH2 Domain (Class 2) ……………………………….86 Table 44. Predicted Binding Partners of the Blnk-SH2 Domain (Class 1) ………………………….……….87 viii Table 45. Predicted Binding Partners of the Blnk-SH2 Domain (Class 2) ………………….……………….88 Table 46. Predicted Binding Partners of the Blnk-SH2 Domain (Class 3) ………………………….……….89 Table 47. Predicted Binding Partners of the SLA2-SH2 Domain (Class 1) ………………………………….90 Table 48. Predicted Binding Partners of the SLA2-SH2 Domain (Class 2) ………………………………….91 Table 49. Predicted Binding Partners of the SH2D3A-SH2 Domain (Class 1) …………………………….92 Table 50. Predicted Binding Partners of the SH2D3A-SH2 Domain (Class 2) …………………………….93 ix List of Figures Figure 1. Src-SH2 domain bound to pYEEI ……………………………………………….………………..………………4 Figure 2. Library used for screening ……………………………………………………….……………………………….9 Figure 3. Screening Scheme ………………………………………………………………….…………………….…………..10 Figure 4. Partial