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Understanding Ubiquitin Recognition and Generating Affinity Reagents

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Understanding Ubiquitin Recognition and Generating Affinity Reagents Ubiquitin Engineering: Understanding Ubiquitin Recognition and Generating Affinity Reagents by Isabel Leung A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Molecular Genetics University of Toronto © Copyright by Isabel Leung 2017 Abstract Ubiquitin Engineering: Understanding Ubiquitin Recognition and Generating Affinity Reagents Isabel Leung Doctor of Philosophy Department of Molecular Genetics University of Toronto 2016 Protein-protein interactions are necessary for virtually all biological processes. There have been tremendous efforts to document the diversity of molecular recognition, and to understand how molecular recognition occurs. The understanding of molecular interaction has also served as the foundation for designing novel protein interactions for use in therapeutics, diagnostics and basic sciences. An attractive system for studying protein-protein interactions is the ubiquitin (Ub) system. Ub is a protein modifier that is combinatorially ligated onto substrate proteins to influence substrate turnover and function. Ub uses a common surface to interact with more than 1000 proteins and plays pivotal roles in cell physiology. Despite the substantial structural information on Ub mediated interactions, there is no clear understanding of how individual Ub residues contribute to Ub’s broad scope of interactions. To address this question, I used affinity enhanced Ub variants (Ubvs) as proxies of native Ub in saturation scanning. Using saturation scanning, I studied the interactions between Ubvs and two Ub specific proteases (USP), USP2 and USP21, and elucidated a common functional epitope that is critical for USP recognition. The functional epitope recognizes USP residues that are conserved among the human USP family, suggesting it may make functional contributions in many other USP interactions. While ii the functional epitopes for Ub binding to USP2 and USP21 share many identical residues, there exist distinct clusters of residues that are responsible for gaining high affinity and specificity to either USP2 or USP21. In this thesis, I also describe the usage of Ub as an alternative scaffold for generating affinity reagents. I developed a novel Ub phage library for the generation of Ubvs to targets of interest. I present validation data on Ubvs selected to bind mammalian cell surface receptor, Her3, and an intracellular protein, Grb2, showing that Ubvs can be used like conventional antibodies in cell biological experiments and can be utilized to antagonize cell signaling within a cell. My work demonstrates that protein-engineering approaches can reveal novel aspects of Ub biology and generate novel affinity reagents to probe intracellular signaling pathways. iii Acknowledgments First and foremost, I thank my parents for leaving behind their established lives in Hong Kong and immigrating to Canada so that my brother and I can have a better future. Without their sacrifices, this PhD degree would likely have been impossible. I sincerely thank my supervisor, Sachdev Sidhu, for taking a chance on me. Not only have I learnt the art of doing science from him, but I also learnt from him the meaning of hard work, determination and most of all what it means to have a vision. I thank my committee members Jason Moffat and James Ellis for their scientific insights and support as well. This degree would have been far less enjoyable without the help from the fun loving, past and present colleagues of the Sidhu lab: Andreas Ernst, Rachel Hanna, Amandeep Gakhal, Nish Patel, Linda Beatty, Esther Lau, Rashida Williams, Joan Teyra, Maryna Gorelik, Lia Cardarelli, Jaspal Singh, Max London, Kristin Kantautus, Natasha Pascoe, Valencio Salema, Moshe Ben-David, Johan Nilvebrant, Jan Tykvart, Gianluca Veggiani, Wei Ye, Alia Pavlenco, Lori Moffat and Danielle Carranza. Finally, I owe a big part of this degree to my fiancé and soon to be husband, Mike Eastwood, for pushing me to the finish line. I will forever be grateful for his, sometimes feeble, attempts to put a smile on my face when times were tough. Thanks for putting up with me. Finally, special shout outs to my friends - Leena Baker, Praan Misir, Tina Shum, and Paul Lee. Thank you. iv Table of Contents Abstract ................................................................................................................................................... ii Acknowledgments ................................................................................................................................ iv Table of Contents .................................................................................................................................. v List of Tables ...................................................................................................................................... viii List of Figures ....................................................................................................................................... ix List of Abbreviations ........................................................................................................................... xi 1 Introduction ..................................................................................................................................... 1 1.1 Protein-Protein Interactions ............................................................................................................... 1 1.1.1 Affinity and Specificity ................................................................................................................................ 1 1.1.2 Binding mechanisms and their effects on affinity and specificity .................................................. 3 1.2 Characteristics of protein binding surfaces ..................................................................................... 6 1.2.1.1 Amino Acid Composition .................................................................................................................... 6 1.2.1.2 Hot spots .................................................................................................................................................... 7 1.2.2 Studying PPIs ................................................................................................................................................... 9 1.2.2.1 Phage Display .......................................................................................................................................... 9 1.2.2.2 Applications of phage display in PPI studies .............................................................................. 11 1.3 Protein Engineering of Affinity Reagents ..................................................................................... 13 1.3.1 Antibodies ....................................................................................................................................................... 14 1.3.1.1 Antibody Limitations .......................................................................................................................... 15 1.3.2 Alternative Scaffolds ................................................................................................................................... 16 1.3.3 In vitro display systems for affinity reagent engineering ................................................................ 17 1.3.4 Examples of Successful Alternative Scaffolds ................................................................................... 19 1.3.4.1 Affibodies ................................................................................................................................................ 19 1.3.4.2 DARPins .................................................................................................................................................. 20 1.3.4.3 Monobodies ............................................................................................................................................ 20 1.3.4.4 Anticalins ................................................................................................................................................ 21 1.3.5 Practical considerations for scaffold engineering .............................................................................. 22 1.4 Ubiquitin .............................................................................................................................................. 23 v 1.4.1 Ub in biology and disease .......................................................................................................................... 23 1.4.2 The Ub fold ..................................................................................................................................................... 26 1.4.3 Ub Interactions ............................................................................................................................................... 27 1.5 Thesis Overview .................................................................................................................................. 29 2 Saturation Scanning of Ubiquitin Variants reveals a common hot spot for binding to USP2 and USP21 ........................................................................................................................... 31 2.1 Statement of Contribution ................................................................................................................ 31 2.2 Introduction ........................................................................................................................................
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