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Sequence Specificity of BUZ, PDZ, SH2, and Tandem BRCT Domains

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Sequence Specificity of BUZ, PDZ, SH2, and Tandem BRCT Domains Sequence Specificity of BUZ, PDZ, SH2, and Tandem BRCT 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 Ryan Hard, B.S. Ohio State Biochemistry Program ***** The Ohio State University 2013 Dissertation Committee: Professor Dehua Pei, Advisor Professor Michael Ibba Professor Jennifer Ottesen Copyright by Ryan Hard 2013 ABSTRACT Src Homology 2 (SH2), Post-Synaptic Density-95/Discs Large/Zonula Occludens-1 (PDZ), Binder of Ubiquitin Zinc Finger (BUZ), and BRCA1 C-terminal (BRCT) domains are short peptide-binding modules that recognize different types of peptide motifs within their protein binding partners. Determination of the type of peptide motifs preferred by each domain would lead to a better understanding of the in vivo role of each domain, including which protein(s) they recognize and how they recognize them. To determine the binding specificity of these domains, we constructed combinatorial one-bead-one-compound (OBOC) peptide libraries and screened them against each domain, sequenced positive hits from the screens by partial Edman degradation/mass spectrometry, and sorted the binding sequences into recognizable patterns. We used in vitro peptide binding assays, site-directed mutagenesis, and pull-down assay techniques to validate the screening results and help understand the nature of the protein-peptide interactions. The SH2 domains of PLCγ1 (both SH2 domains) and TSAd were screened against phosphotyrosine (pY)-containing OBOC peptide libraries. The SH2 domain of TSAd selected for only one class of sequences, while the other SH2 domains each selected multiple classes of ligands. Generally, the SH2 domains showed selectivity for residues at the +1, +2, and +3 positions (relative to pY) and not for residues N-terminal to pY. PDZ and BUZ domains recognize their protein binding partners at the C-terminus, specifically requiring the free carboxylate group for efficient binding. In order to probe ii the binding specificity of these C-terminal binding domains, we constructed spatially segregated OBOC peptide libraries, where peptides in the exterior of each bead presented a free C-terminus and could therefore interact with the PDZ and BUZ domains, while peptides in the bead interior presented a free N-terminus so that they could be sequenced using partial Edman degradation and mass spectrometry. Using this type of library, the C-terminal sequence specificity of the PDZ domains of Tiam1 and Tiam2 and the BUZ domains of Ubp-M and HDAC6 were examined. Each PDZ and BUZ domain selected for multiple classes of binding sequences. In vitro peptide binding studies were used to verify the BUZ/PDZ-peptide interactions, including ones with a Tiam1 mutant that helped to illustrate the basis of PDZ domain ligand affinity and specificity. Furthermore, the BUZ domain of Ubp-M was used in a pull-down assay of a protein containing a C-terminus matching one of its consensus binding sequences, which further validated the screening results. Tandem BRCT domains recognize their protein binding partners either at internal or C-terminal peptide motifs. In either case, they usually require at least one phosphoserine (pS) or phosphothreonine (pT) residue to bind with high affinity. In order to determine the phosphopeptide binding specificity of the tandem BRCT domains, we constructed two pS/pT OBOC libraries. One pS/pT OBOC library, like the libraries used to study the PDZ and BUZ domains, presented peptides with free C-termini on the library bead surfaces and contained encoding peptides with free N-termini in the bead interior. A second library was constructed where the peptides were presented in the normal N- terminal to C-terminal direction on both the interior and exterior portions of the library beads. All 16 known human tandem BRCT domains (or their closel related mouse orthologs) were screened against both phosphopeptide libraries, with 8 of the 16 domains iii giving either well-defined or general consensus binding motifs. The C-terminal BRCT repeat of PTIP was found to bind to a novel dual phosphoserine motif. The remainder of the domains either failed to show binding selectivity or showed binding selectivity in the peptide library screens but failed to bind to resynthesized peptides in solution. iv DEDICATION Dedicated to my family v ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Dehua Pei, for his guidance throughout my career at Ohio State. His advice on how to think about and practice science will help guide me in my future career. I would also like to thank my committee members for their guidance on my candidacy exam and dissertation defense. I also would like to thank Dr. Pei Zhou, Dr. Ernesto Fuentes, and Dr. Junjie Chen for their collaborations. I am grateful to my current and former labmates for their assistance in my career at Ohio State. I am especially grateful to Dr. Sang Hoon Joo for synthesizing the library I first worked with and to Dr. Tao Liu for his advice on protein purification and peptide library synthesis. I would also like to thank Dr. Pauline Tan for her guidance on a variety of different subjects. Finally, I would like to thank my parents and grandparents for their support and encouragement over the years. My parents pushed me to work hard, which was critical to my academic success. I would also like to thank them for their financial support, which allowed me to focus more on my academic career and less on my basic needs. vi VITA May 2007 ……………………………... B.S. Biochemistry Ohio Northern University 2007-2013 ……………………………. Graduate Teaching and Research Associate The Ohio State University PUBLICATIONS 1. Hard, R., Liu, J., Shen, J., Zhou, P., and Pei, D. “HDAC6 and Ubp-M BUZ domains recognize specific C-terminal sequences of proteins”, Biochemistry 2010, 49, 10737-10746. 2. Shepherd, T., Hard, R., Murray, A., Pei, D., and Fuentes, E. “Distinct ligand specificity of the Tiam1 and Tiam2 PDZ domains”, Biochemistry 2011, 50, 1296-1308. 3. Zhang, Y., Zhang, J., Yuan, C., Hard, R., Park, I., Li, C., Bell, C., and Pei, D. “Simultaneous Binding of Two Peptidyl Ligands by a Src Homology 2 Domain”, Biochemistry 2011, 50, 7637-7646. FIELDS OF STUDY Major Field: Biochemistry vii TABLE OF CONTENTS Page Abstract ......................................................................................................................... ii Dedication ..................................................................................................................... v Acknowledgements ....................................................................................................... vi Vita ................................................................................................................................ vii List of Tables ................................................................................................................ xii List of Figures ............................................................................................................... xv List of Abbreviations .................................................................................................... xviii Chapter 1: Introduction ............................................................................................... 1 1.1 Modular Protein Domains .................................................................................... 1 1.2 PDZ Domains....................................................................................................... 1 1.2.1 Structure of PDZ Domains and Mechanism of C-Terminal Ligand Recognition ............................................................................................... 2 1.2.2 Function of PDZ Domains ..................................................................... 3 1.3 BUZ Domains ...................................................................................................... 4 1.3.1 Structure of BUZ Domains and Mechanism of Ubiquitin Recognition ... 4 1.3.2 Function of BUZ Domains..................................................................... 5 1.4 SH2 Domains ....................................................................................................... 6 1.4.1 Structure of SH2 Domains and Mechanism of Ligand Recognition ........ 6 1.4.2 Function of SH2 Domains ........................................................................ 7 1.5 Tandem BRCT Domains..................................................................................... 8 1.5.1 Structure of Tandem BRCT Domains and Mechanism of Ligand Recognition ............................................................................................. 9 1.5.2 Function of Tandem BRCT Domains .................................................... 12 1.6 Methods to Determine the Sequence Specificity of PDZ, BUZ, SH2 and Tandem BRCT Domains.................................................................................... 14 1.6.1 Biological Library Techniques................................................................. 15 1.6.1.1 Yeast Two-Hybrid System ......................................................... 15 1.6.1.2 Phage Display ............................................................................ 16 1.6.1.3 lacI Repressor ............................................................................ 17 1.6.1.4 FRET-Based Screening Assay ................................................... 18 1.6.1.5 Co-immunoprecipitation/Pull-Down Assays ............................
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