Chemical and Biochemical Studies of Ubiquitin Conjugation Machinery by Renuka K. Pandya B.A. Biology Cornell University, 2004 SUBMITTED TO THE DEPARTMENT OF BIOLOGY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTORATE OF PHILOSOPHY IN BIOLOGY AT THE MASSACHUSETTS INSTITUTE OF TECHNOLOGY JUNE 2010 © 2010 Massachusetts Institute of Technology All rights reserved Signature of Author………………………………………………………………………………… Renuka K. Pandya Department of Biology May 24, 2010 Certified by………………………………………………………………………………………… Hidde L. Ploegh Member, Whitehead Institute Professor of Biology Thesis Supervisor Accepted by………………………………………………………………………………............... Tania A. Baker Professor of Biology Co-Chair, Biology Graduate Committee 1 Chemical and Biochemical Studies of Ubiquitin Conjugation Machinery by Renuka K. Pandya Submitted to the Department of Biology on May 24, 2010 in Partial Fulfillment of the Requirements for the Degree of Doctorate of Philosophy in Biology at the Massachusetts Institute of Technology Abstract The post-translational modification of proteins is a major mechanism employed in eukaryotic cells to expand the functional diversity of the proteome. Covalent modification of amino acid side chains confers new or altered functionality to the modified protein by creating new recognition surfaces on the protein for the interaction of nucleic acids or other proteins, modulating enzymatic activity, or altering cellular localization or half-life. The post- translational modification of proteins with ubiquitin (Ub) is an important mechanism of regulating protein function. Ub is a 76-residue protein that is primarily attached to lysine residues in target proteins through an enzymatic cascade catalyzed by E1, E2, and E3 enzymes. Ub conjugation is important for fundamental cellular processes, including transcription, DNA repair, endocytosis, apoptosis, and signal transduction. Ub conjugation is reversible. Proteases termed deubiquitinating enzymes (DUBs) function to remove Ub from target proteins. Genome sequencing efforts have uncovered the existence of many predicted enzymes with unknown function. Many enzymes have been assigned function based on sequence homology to proteins with known function without confirmation of enzymatic activity. A powerful chemical approach to determine enzyme function from a complex mixture of proteins is activity-based protein profiling. This method makes use of chemical probes that are active site-directed for the assignment of function to proteins. We describe here the design and generation of an expanded set of Ub-based chemical probes with which we identified and recovered E1, E2, and E3 Ub ligases from cell lysates. Furthermore, we describe the biochemical and structural characterization of the catalytic domain of one E3 Ub ligase we recovered, HUWE1, and the identification of a structural element within the catalytic domain of HUWE1 that modulates its activity. Finally, we discuss a protein engineering method that we are applying to the HUWE1 catalytic domain to understand how the conformational flexibility of this domain is important to its function. Thesis Supervisor: Hidde L. Ploegh Title: Member, Whitehead Institute; Professor of Biology 2 TABLE OF CONTENTS Abstract……………………………………………………………………………………. 2 Acknowledgements……………………………………………………………………….. 6 CHAPTER 1: Introduction……………………………………………………………….9-56 Introduction to ubiquitin conjugation 10 Ubiquitin-like modifiers 13 Ubiquitin activation and conjugation enzymes and ligases 16 Ub/Ubl activating enzyme (E1) 17 Ub/Ubl conjugating enzyme (E2) 23 Ubiquitin ligases (E3) 28 RING E3 ligases 28 HECT E3 ligases 31 Polyubiquitin chain assembly 32 Deubiquitinating enzymes/proteases 37 Activity-based protein profiling (ABPP) 39 HUWE1 E3 Ub ligase 44 Conclusions 46 References 47 CHAPTER 2: Ubiquitin C-terminal electrophiles are activity-based probes for identification and mechanistic study of ubiquitin conjugating machinery……………57-92 Abstract 58 Introduction 59 Results and Discussion 61 Synthesis and characterization of second generation HAUb-electrophilic probes 61 Profiling and identification of enzymes modified by newly synthesized HAUb-electrophilic probes in EL-4 and HMLE cell lysate 62 HAUb-electrophilic probes are activity-based probes for E3 Ub ligases 66 Multiple cysteines in ARF-BP1 form thioesters with Ub 68 Conclusions 71 Methods 72 Acknowledgements 76 References 77 Figure and Table Legends 81 Figures 85 CHAPTER 3: A structural element within the HUWE1 HECT domain modulates self-ubiquitination and substrate ubiquitination activities……………………………..93-126 Abstract 94 Introduction 95 3 Results 96 Structure of the HUWE1 HECT domain 96 Catalytic activity of the HECT domain 99 Thioester formation in the HECT domain 100 Substrate ubiquitination catalyzed by the HECT domain 101 Catalytic activity of the C4341A mutants 102 Discussion 103 Experimental procedures 107 Acknowledgements 111 References 112 Figure Legends 114 Figures 118 CHAPTER 4: Sortase-catalyzed circularization of HUWE1 HECT domain………...127-150 Abstract 128 Introduction 129 Results and Discussion 131 Circularization of HUWE1-sortag1 131 Biochemical characterization of circular HUWE1-sortage1 132 Experimental procedures 138 References 142 Figure Legends 144 Figures 147 CHAPTER 5: Future Directions………………………………………………………...151-168 Summary 152 Activity-based inhibitors of Ub conjugation enzymes 153 Development of activity-based protein probes for Ub conjugating machinery 153 Structural insights into Ub conjugation using activity-based inhibitors 155 Improvements to Ub C-terminal electrophilic probes 156 Identification of Ubl conjugation machinery using activity-based inhibitors 158 Role of non-covalent interactions during Ub transfer 159 Probing conformational flexibility of HECT domains using sortase-mediated protein engineering 159 Physiological functions of E3 Ub ligases 160 Identification of physiologically relevant E2-E3 pairs 161 Identification of E3 ligase substrates 162 Conclusions 163 References 165 APPENDIX A: Supplementary information for “Ubiquitin C-terminal electrophiles are activity-based probes for identification and mechanistic 4 study of ubiquitin conjugating machinery……………………………………………...169-188 Supplementary Methods 170 Supplementary References 174 NMR characterization 176 LC-MS characterization 179 Supplementary Figure Legends 180 Supplementary Figures 181 APPENDIX B: Supplementary Information for “A structural element within the HUWE1 HECT domain modulates self-ubiquitination and substrate ubiquitination activities………………………………………………………………......189-194 Supplementary Figure Legends 190 Supplementary Figures 192 5 Acknowledgements Many people have shaped my time as a graduate student at MIT. I thank my advisor, Hidde Ploegh, for encouraging me to reach far, think big, and put my best foot forward. He entrusted me to pursue my interests with enthusiasm tempered by skepticism from the day I spoke with him. My committee members, Mike Yaffe and Tania Baker, have also been instrumental in fostering my learning throughout the years, and I appreciate their valuable input during both committee meetings and informal conversations. I thank Thomas Schwartz and Wade Harper for serving on my thesis defense committee. A special thanks to Thomas Schwartz and James Partridge, with whom I was fortunate to collaborate. I learned a great deal from just a short walk across the street to the Schwartz lab. The Ploegh lab is full of bright individuals who have contributed to my thesis through numerous conversations, by sharing advice, by looking at data, or through friendship. Thanks to Kerry Love, with whom I worked for two years, for both scientific conversations and laughter, and to Robert Miller, for his good cheer, and for making many things possible on short notice. Thanks also to Shahram Misaghi, Greg Korbel, Ferenc Reinhardt, Eric Spooner, Tom DiCesare, Christian Schlieker, Jadyn Damon, Nick Yoder, Max Popp, John Antos, and Clarissa Lee. I’m lucky to have some wonderful graduate school friends, Jane Kim and Wendy Lam. Thank you for your friendship, support, camaraderie, and commiseration. I thank my family: my big brother Naveen Sastry, whom I’ll always look up to, and my parents, K.V. and Sesha Sastry. Their love and support is constant and tangible. Finally, I thank my husband, Ankur Pandya. I can’t imagine where I’d be without you and the joy you bring me every day. Thank you for believing in me. 6 For my parents 7 8 Chapter 1: Introduction CHAPTER 1: INTRODUCTION 9 Chapter 1: Introduction Introduction Post-translational modification of proteins is one of two major mechanisms employed by eukaryotic cells to expand the functional diversity of the proteome. This mechanism, along with RNA splicing, increases the number of protein variants in the cell by one to two orders of magnitude over those encoded in the genome (Walsh, Garneau-Tsodikova et al. 2005; Walsh 2006). Covalent modification of amino acid side chains increases their heterogeneity and imparts new or altered functionality to the modified protein by creating new recognition surfaces on the protein for the interaction of nucleic acids or other proteins, modulating enzymatic activity, or altering cellular localization or half-life. Post-translational modifications are utilized in all three major evolutionary lineages of life; however, the range of modifications and frequency of occurrence in eukaryotes far exceeds that in prokaryotes and archaea. Here, we discuss the covalent modification of proteins with ubiquitin