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I SRC HOMOLOGY 2 DOMAIN PROTEINS SRC HOMOLOGY 2 DOMAIN PROTEINS BINDING SPECIFICITY: FROM COMBINATORIAL CHEMISTRY TO CELL-PERMEABLE INHIBITORS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Anne-Sophie M. Wavreille, M. S. The Ohio State University 2006 Dissertation Committee: Professor Dehua Pei, Adviser Approved by Professor Ross E. Dalbey Professor Thomas J. Magliery Professor Susheela Tridandapani Adviser Chemistry Graduate Program i ABSTRACT Protein-protein interactions form the molecular basis of a wide variety of cellular processes. A large fraction of these interactions are mediated by small modular domains, which bind to short peptide motifs in their partner proteins. However, for the vast majority of these modular domains, their binding specificity and interacting partners remain unknown. This work presents a chemical/bioinformatic approach to the identification of the binding proteins of the Src Homology 2 domain (SH2). First, a combinatorial phosphotyrosyl (pY) peptide library was screened to determine the amino acid preferences at the pY+4 to pY+6 positions for the four SH2 domains of protein- tyrosine phosphatases SHP-1 and SHP-2. The screening results were confirmed by surface plasmon resonance analysis, stimulation assays and in vitro pull-down experiments. This study reveals that binding of a pY peptide to the N-SH2 domain of SHP-2 is greatly enhanced by a large hydrophobic residue at the pY+4 and/or pY+5 positions, whereas binding to SHP-1 N-SH2 domain is enhanced by either hydrophobic or positively charged residues at these positions. Similar residues at the pY+4 to pY+6 positions are also preferred by SHP-1 and SHP-2 C-SH2 domains, although their ii influence on the overall binding affinities is much smaller compared with the N-SH2 domains. Second, our chemical/bioinformatic approach was applied to identify the binding proteins of tensin. Another pY peptide library was screened against the tensin SH2 domain to determine the peptide motifs that bind to this domain. The peptide motifs were employed to search protein databases for potential tensin-binding proteins, which were subsequently confirmed (or disproved) by in vitro pull-down and co- immunoprecipitation assays. This procedure identified phosphoinositide-dependent kinase-1 (PDK-1) and downstream of tyrosine kinase 2 (Dok-2) as novel tensin-binding proteins. In addition, a cell-permeable pY peptide was designed as tensin SH2 domain inhibitor, which caused the disruption of actin filaments in NIH 3T3 cells. Third, the sequence specificity of other SH2 domains (c-Src, Grb2, Syk, ZAP-70 and SLAP) were established. Finally, our strategy was applied to other modular domains: chromodomains, another type of library: a cyclic peptide library and a different screening: a live cell- binding assay. iii Dedicated to my parents iv ACKNOWLEDGMENTS I wish to thank my advisor, Dr. Dehua Pei, for his fantastic enthusiasm and his great intellectual support. His broad scientific knowledge, along with his way of teaching and lab managing gave me one of the best training I could ever have. I will use them as a model throughout my career. I would like to thank my senior labmates, Dr. Michael Sweeney, Dr. Kiet Nguyen, Dr. Junguk Park, and Dr. Diana Imhof. You brought me a tremendous professional support, but it is especially your high spirits and your friendship that made me so happy to work with you all. I also wish to thank my current labmates, Dr. Qing Xiao, Jing Zhang, Mathieu Garaud, Sang Hoon Joo, Amit Thakkar, and particularly Yanyan Zhang for their stimulating discussions. I am endebted to Dr. Susheela Tridandapani and her whole team, Dr. Jonathan Butchar, Dr. Latha Ganesan, Dr. Murugesan Rajaram, Kishore Parsa, and Trupti Joshi for teaching me the mammalian cell assays and providing me access to their equipment. I am eternally grateful to my parents and my brothers for their formidable support and encouragement in spite of the geographical distance between us. Finally, I would like to say a special thank you to Eric, for his precious moral support. v VITA 2000-2001………………...Technician, Oleochemistry Department, Cognis-Henkel, Düsseldorf, Germany. 2002………………………M.S. Chemistry and Process Engineering, CPE Lyon, France. 2001-2006………………...Graduate Teaching and Research Associate, The Ohio State University PUBLICATIONS 1. Wavreille Anne-Sophie, Pei Dehua. A chemical approach to the identification of tensin-binding proteins. ACS Chemical Biology (2006) In press. 2. Thakkar Amit, Wavreille Anne-Sophie, Pei Dehua. A traceless capping agent for peptide sequencing by partial Edman degradation and mass spectrometry. Analytical Chemistry (2006) 78, 5935-5939. 3. Imhof Diana*, Wavreille Anne-Sophie*, May Andreas, Zacharias Martin, Tridandapani Susheela, Pei Dehua. Sequence specificity of SHP-1 and SHP-2 SH2 domains: Critical roles of residues beyond the pY+3 position. J. Biol. Chem. (2006) 281, 20271-20282. 4. Qin Chuanguang, Wavreille Anne-Sophie, Pei Dehua. Alternative mode of binding to phosphotyrosyl peptides by Src Homology-2 domains. Biochemistry (2005), 44 (36), 12196-12202. 5. Sweeney Michael C., Wavreille Anne-Sophie, Park Junguk, Butchar Jonathan P., Tridandapani Susheela, Pei Dehua. Decoding protein-protein interactions through combinatorial Chemistry: sequence specificity of SHP-1, SHP-2, and SHIP SH2 domains. Biochemistry (2005), 44 (45), 14932-14947. vi 6. Zhu Jinge, Dizin Eric, Hu Xubo, Wavreille Anne-Sophie, Park Junguk, Pei Dehua. S-Ribosylhomocysteinase (LuxS) is a mononuclear iron protein. Biochemistry (2003), 42 (16), 4717-4726. 7. Prinz Daniela, Herault David, Wavreille Anne-Sophie. Oil phases containing silanes for the preparation of cosmetic emulsions. PCT Int. Appl. (2003), 28 pp. *: Equal contribution FIELDS OF STUDY Major Field: Chemistry vii TABLE OF CONTENTS Page Abstract……………………………………………………………………………………ii Dedication…………………………………………………………………………….......iv Acknowledgments………………………………………………………………………....v Vita………………………………………………………………………………………..vi List of Tables…………………………………………………….....………...………….xii List of Figures...................................................................................................................xiv List of Abbreviations.......................................................................................................xvii Chapters: 1. Introduction..............................................................................................................1 1.1 Protein domains and the example of SH2 domain..........................................1 1.2 Methods for the determination of SH2 domain sequence specificity.............4 1.2.1 Affinity purification / pool sequencing...............................................4 1.2.2 Phage display......................................................................................6 1.2.3 Positional scanning.............................................................................9 1.2.4 Chemical microarray.........................................................................11 1.3 Methods for the identification of protein partners of the SH2 domains.......13 1.3.1 Co-immunoprecipitation / protein pull-down...................................13 1.3.2 Two-hybrid system...........................................................................16 1.3.3 Computational modeling...................................................................19 1.3.4 Combinatorial peptide library...........................................................20 2. Extended sequence specificity of SHP-1 and SHP-2 SH2 domains......................26 2.1 Introduction...................................................................................................26 2.2 General experimental procedures.................................................................29 2.2.1 Library synthesis on solid phase.......................................................29 2.2.2 SH2 domain constructs.....................................................................31 2.2.3 Library screening..............................................................................33 2.2.4 Peptides sequencing..........................................................................34 2.2.5 Individual peptides synthesis............................................................35 2.2.6 Stimulation of phosphatase activity..................................................36 viii 2.2.7 Determination of dissociation constants by Surface Plasmon Resonance (SPR)...........................................................................37 2.2.8 In vitro peptide pull-down assay.......................................................38 2.2.9 In vitro co-immunoprecipitation assay in the presence of competing peptides.............................................................................................39 2.2.10 Site-directed mutagenesis of FcγRIIb to enhance SHP-1 and SHP-2 binding..............................................................................................40 2.2.11 Database search.................................................................................41 2.3 Results...........................................................................................................41 2.3.1 Library design...................................................................................41 2.3.2 Screening results...............................................................................42 2.3.3 Affinity measurements by surface plasmon
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