SEQUENCE SPECIFICITY OF SRC HOMOLOGY 2 DOMAINS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Pauline H. Tan, B. S. Graduate Program in Chemistry The Ohio State University 2011 Committee: Professor Dehua Pei, Advisor Professor Jennifer J. Ottesen Professor Karin Musier-Forsyth Copyright by Pauline H. Tan 2011 ABSTRACT Src-homology domains are small modular domains that recognize phosphotyrosine-containing proteins and couple activated protein kinases to intracellular signaling pathways. Since they often have overlapping functions, their SH2 domains often compete for binding to the same pY proteins. Determining their sequence specificities will help identify target proteins. Consequently, this will help understand the molecular basis for their cellular functions. Twenty-six kinase SH2 domains were screened against a phosphotyrosyl (pY) peptide library, and positive beads were sequenced by partial Edman degradation and mass spectrometry. The data revealed that the kinase family SH2 domains selected a class of pY peptides consisting of mostly hydrophilic and hydrophobic residues at the pY+1 and pY+3 positions, respectively. After validating their binding, the literature was searched to find known SH2 targets and their pY motifs. Seventeen SH2 domains from several different protein families were also purified, screened, and sequenced to determine their binding motifs. The majority of the SH2 domains had high selectivity at the pY+3 or pY+1 position with a few selecting for multiple peptide classes. For example Vav1 and Vav2 SH2 domains selected for three classes of peptides. These minor classes of peptides may be motifs of new protein targets that have not been identified yet. Some SH2 domains such as the Grb7 family, HSH2D, ii and Vav family had high selectivity of Asn at the +2 position but little selectivity at other positions. More subtle differences were observed between protein families at certain positions. For instance, SH2 domains from a family of GTPase signaling proteins preferred Pro at the +3 position, while SH2 domains from the PIK3 family preferred norleucine at the +3 position. Genetic disorders such as Noonan’s syndrome and hematologic disorders such as juvenile myelomonocytic leukemia are caused by mutations in the SHP2 (PTPN11) gene. The mutations are mainly located in the SH2 binding cleft or in the NSH2/PTP interface. Mutations in the SH2 binding cleft may alter sequence specificity, resulting in new potential binding partners. Identifying novel binding partners may help lead to a better understanding of the complex mechanism of these disorders. Five SHP2 SH2 mutants were screened against a combinatorial phosphotyrosyl (pY) peptide library, and positive hits were sequenced. Only the T52S SH2 mutant exhibited a specificity switch from small to large branched hydrophobic residues at the +1 position. The T42A and L43F SH2 mutants exhibited increased binding affinities. In contrast, the specificities and binding of E76K and E139D SH2 mutants remained unchanged. After examining the crystal structure generated by PyMOL, altered specificity involved a substitution of a bulky Thr with a smaller Ser residue allowing more space for larger branched residues. The T52S consensus was entered into a protein database to search for new protein targets that bind to the mutant only. Twenty-six potential targets resulting from the database search were identified for the T52S mutant. iii DEDICATION Dedicated to my family iv ACKNOWLEDGMENTS I would like to thank my advisor Dr. Dehua Pei for his constant intellectual guidance and encouragement throughout these years. His dedication to science will serve as guide for me to achieve in my career. My experience in the Pei lab has provided me many opportunities to grow not only as a scientist but also as a person. I also wish to thank my committee members for guiding me during my candidacy and dissertation defense. I am deeply indebted to my senior labmates Dr. Anne-Sophie Wavreille and Dr. Yanyan Zhang, for their guidance on the projects and answering many questions. I would also like to thank my colleagues Dr. Qing Xiao, Dr. Amit Thakkar, Dr. Tao Liu, Dr. Xianwen Chen, Nick Selner, Tiffany Meyer, and Andrew Kunys, and the rest of the Pei lab members, past and present, for their support, friendship and intellectual discussions at lunchtime. Finally I wish to thank my parents and sister for their unconditional love, moral support, and encouragement. I would like to thank my parents for teaching me to work hard and achieve to my best ability. I also want to thank my sister Stephanie for her friendship and support throughout these years. Without hard work and family support, I would not be where I am now. v VITA June 2005 .......................................................B. S. in Chemistry & Biochemistry The Ohio State University 2005-2011 ......................................................Graduate Teaching and Research Associate The Ohio State University PUBLICATIONS 1. Chen, X., Tan, P., Zhang, Y. and Pei, D., “On-bead screening of combinatorial libraries: reduction of nonspecific binding by decreasing surface ligand density”, J. Comb. Chem. 2009, 11, 604-611. FIELDS OF STUDY Major Field: Chemistry vi TABLE OF CONTENTS Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iv Acknowledgments............................................................................................................... v Vita ..................................................................................................................................... vi List of Tables .................................................................................................................... xii List of Figures ................................................................................................................... xv List of Abbreviations ...................................................................................................... xvii Chapter 1 Introduction ........................................................................................................ 1 1.1. SH2 Domains .......................................................................................................... 1 1.2. Other Methods to determine the SH2 specificity .................................................... 5 1.2.1 Solution phase pooled library ............................................................................ 5 1.2.2. Microarray library (Oriented peptide array library (OPAL)) ........................... 6 1.2.3. One bead one compound library (OBOC) ........................................................ 7 1.2.4. Phage display .................................................................................................. 12 1.2.5. Two-hybrid systems ....................................................................................... 13 vii Chapter 2 Specificity of kinase SH2 domains .................................................................. 15 2.1. Introduction ........................................................................................................... 15 2.2. Experimental Procedures....................................................................................... 19 2.2.1 Materials .......................................................................................................... 19 2.2.2. Synthesis of the pY library and individual peptides ....................................... 20 2.2.3. SH2 constructs ................................................................................................ 22 2.2.4. Protein expression and purification ................................................................ 23 2.2.5. SH2 protein labeling ....................................................................................... 24 2.2.6. pY library screening ....................................................................................... 25 2.2.7. Partial Edman degradation.............................................................................. 26 2.2.8. Synthesis of individual pY peptides ............................................................... 27 2.2.9. Determination of dissociation constants by SPR ............................................ 28 2.2.10. Determination of dissociation constants by fluorescence polarization ........ 29 2.3. Results ................................................................................................................... 29 2.3.1. pY library synthesis and screening ................................................................. 30 2.3.2. General specificity of the kinase family SH2 domains .................................. 31 2.3.3. Sequence specificity of Src family kinase SH2 domains ............................... 31 2.3.4. Abl1 and Abl2 SH2 domains .......................................................................... 44 2.3.5. Csk and MATK SH2 domains ........................................................................ 48 viii 2.3.6. Fes and Fer SH2 domains ............................................................................... 51 2.3.7. Tec family kinase SH2 domains ....................................................................