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Structure Determination and Biochemical Characterization of Novel Human Ubiquitin-Like Domains

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Structure Determination and Biochemical Characterization of Novel Human Ubiquitin-Like Domains STRUCTURE DETERMINATION AND BIOCHEMICAL CHARACTERIZATION OF NOVEL HUMAN UBIQUITIN-LIKE DOMAINS. by Ryan Steven Doherty A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Medical Biophysics University of Toronto © Copyright by Ryan Steven Doherty 2015 STRUCTURE DETERMINATION AND BIOCHEMICAL CHARACTERIZATION OF NOVEL HUMAN UBIQUITIN-LIKE DOMAINS Ryan Steven Doherty Doctor of Philosophy Department of Medical Biophysics University of Toronto 2015 Abstract The ubiquitin fold acts as a signaling modulator associated with regulating, trafficking, and degrading proteins. The human genome encodes 398 ubiquitin-like domains (UBLs), of which a couple dozen may act as covalent modifiers. Ubiquitin and ubiquitin-like domains have been implicated in a number of malignancies, neuromuscular disorders, neurodegenerative disorders and other human illnesses. Identifying the structural effects of sequence variations between different ubiquitin-like homologues will provide insight into their varied functional pathways, since the role of ubiquitin-like modifiers is typically mediated by protein-protein interactions. Structure determination and analyses of ubiquitin-like homologues facilitates residue mapping and comparative analysis of protein-protein interaction sites, which provide insight into the many roles that ubiquitin-like homologues play in cellular processes. The aim of this thesis was to develop a framework through which complete structural coverage of all human ubiquitin-like domains could be achieved. To accomplish this, I defined the human ubiquitin-like fold family, identified ubiquitin- like domain constructs amenable for NMR structure determination, solved two structures ii (NFATc2IP & ubiquilin-1) and characterized associated binding partners, and created a data resource for human ubiquitin-like domains that enables clustering and associating protein structures with physicochemical features and cellular function. I also collaborated with the North- East Structural Genomics consortium (NESG) and the Structural Genomics Consortium (SGC), through which the molecular structures of 17 ubiquitin-like domains were determined using nuclear magnetic resonance (NMR) experiments and X-ray crystallography. Comparative analysis of structurally characterized ubiquitin-like folds revealed potential interaction partners with regions similar to known ubiquitin and SUMO interacting domains. Potential interaction partners for NFATC2IP and ubiquilin-1 were validated experimentally using NMR titration experiments. Comparative analysis of structural features of all ubiquitin-like homologues facilitates further studies into the mechanisms of the ubiquitylation system, predicted protein- protein interactions, and the identification of functional pathways associated with uncharacterized ubiquitin-like domains. iii Acknowledgements I would like to thank my supervisor Cheryl Arrowsmith for her ongoing support, advice and mentorship over the years. I also appreciate the guidance and knowledge shared by my supervisory committee members: Sirano Dhe-Paganon, Brian Raught, Jane McGlade, and Zhaolei Zhang. I would also like to recognize the efforts and support from members of the Arrowsmith lab, past and present, especially Adelinda Yee, Shili Duan, Scott Houliston, Sasha Lemak, Aleks Gutmanas, Christophe Fares, Yi Sheng, Lilia Kaustov, Bin Wu, Seth Chitayat, Sampath Srisailam, Murthy Karra, Jonathan Lukin, Natalie Nady, Jack Liao, Rob Laister, Melissa Ho, Tony Semesi, and Maite Garcia. This thesis would not have been possible without collaborations. For this reason, I would like to thank Gaetano Montelione, John Everett, Mani Ravichandran, Yufeng Tong, Masoud Vedadi, David Yim and Raymond Hui for their time, resources, feedback and help in key aspects of this project. I would also like to thank members of various University of Toronto communities who have encouraged, supported and worked alongside me throughout this endeavor: Medical Biophysics Graduate Student Association, 89 Chestnut Residence, Massey College, Massey Grand Rounds, and Impact Centre. Finally, I thank my family and friends for their patience, love and understanding. It is to you that I dedicate this thesis. iv Table of Contents Abstract ii Acknowledgements iv Table of Contents v List of Tables x List of Figures xi List of Appendices xiii List of Abbreviations xiv Chapter 1 - Introduction 1 1.1 Overview 1 1.2 Biological Significance of ubiquitin & ubiquitin-like modifiers 2 1.3 Protein Modification & ubiquitin 2 1.4 The ubiquitin Fold 3 1.5 Ubiquitin-like domains (UBLs) 4 1.6 Ubiquitin-like modifiers (UBM) 6 1.7 Ubiquitin-like structural domains 9 1.8 Ubiquitin Conjugation Cascade 9 1.9 Ubiquitin-binding domains & interactions 11 1.9.1 Ubiquitin Interacting Motif (UIM) 11 1.9.2 Coupling of Ubiquitin conjugation to Endoplasmic Reticulum 12 Degradation (CUE) 1.9.3 Ubiquitin-Associated Domain (UBA) 12 1.9.4 Ubiquitin Conjugating Enzyme Variant (UEV) 12 1.9.5 Npl4 Zing Finger Motif (NZF) 13 1.9.6 GGA And Tom1 Domain (GAT) 13 1.9.7 Other Ubiquitin Binding Domains 13 1.9.8 SUMO Interacting Motif (SIM) 13 v Table of Contents (continued) 1.9.9 Diversity among Ubiquitin-Binding Domains 14 1.10 Thesis Overview 15 1.10.1 Identify and obtain near-complete structural coverage of all 15 human ubiquitin-like domains. 1.10.2 Exploring NFATc2IP:NFATc2 & ubiquilin-1:PIN2 15 protein-protein interactions Chapter 2 - The Ubiquitin Fold: Leveraging structural genomics 17 2.1 Summary 18 2.2 Introduction 18 2.3 Methods 21 2.3.1 Identifying human ubiquitin-like domains 21 2.3.2 Validating putative human ubiquitin-like domains 22 2.3.3 Target selection 24 2.3.4 Construct design 25 2.3.5 Sample preparation 26 2.3.6 1H15N-HSQC screening of ubiquitin-like domains 26 2.4 Results & Discussion 27 2.4.1 Identifying unannotated human ubiquitin-like domains 27 2.4.2 Small-Scale Screening 29 2.4.3 Screening by 1H15N-HSQC 29 2.4.4 Structural Coverage - Completing the UBL Phylogenetic Tree 31 2.5 Conclusion 36 Chapter 3 - Solution NMR structure determination of human ubiquitin-like domains 37 in NFATc2IP & ubiquilin-1 3.1 Introduction 38 3.1.1 NFATc2IP 38 3.1.2 Ubiquilin-1 39 vi Table of Contents (continued) 3.1.3 Ubiquitin-like Fold 39 3.2 Experimental Procedures 40 3.2.1 NFATc2IP UBL domain NMR structure determination 40 3.2. 2 Ubiquilin-1 UBL domain NMR structure determination 41 3.2. 3 Comparative analysis of ubiquilin-1, NFATc2IP, 44 ubiquitin & SUMO2 3.2. 4 Protein-protein interaction partner identification 46 3.2. 5 Binding interface analysis 46 3.3 Results & Discussion 47 3.3.1 Structure determination 47 3.3.2 Comparative analysis of ubiquilin-1, NFATc2IP & similar 52 ubiquitin-like modifiers 3.3.2.1 Similar canonical ubiquitin-like modifiers: ubiquitin & SUMO-2 53 3.3.2.2 Structural comparison between ubiquilin-1 & NFATc2IP 53 3.3.2.3 Structural comparison between ubiquilin-1 & ubiquitin 55 3.3.2.4 Structural comparison between NFATc2IP & SUMO2 57 3.3.2.5 Structural differences between NFATc2IP_2nd & SUMO2 58 3.3.3 From Structure to Function: Exploring Protein-Protein Interactions 59 involving ubiquitin-like domains 3.3.3.1 The ubiquitin-Interacting Motif interaction interface 59 3.3.3.2 Putative UIM Interaction Interface: Conserved Amino Acids 62 3.3.3.3 Putative UIM Interaction Interface: Similar Electrostatic 63 Potential Distribution 3.3.3.4 Surveying Known UIM-Binding Partners 64 3.3.3.5 PIN1 – Peptidyl-Prolyl cis/trans Isomerase 67 3.3.3.6 Identifying a putative UIM in PIN1 67 3.3.3.7 Ubiquilin-1 & PIN1 NMR Titration 68 vii Table of Contents (continued) 3.3.3.8 Analysis of the ubiquilin-1 & PIN1 interface 70 3.3.4 Binding-Partner Driven - Structural analysis of the 71 SUMO-Interacting Motif binding interface 3.3.4.1 NFATc2IP Binding Partners 71 3.3.5 SUMO-Interacting Motif 72 3.3.5.1 Identifying putative SIMs in NFATc2 72 3.3.6 NFATc2IP:NFATc2 NMR titration 74 3.3.6.1 Analysis of the NFATc2IP:NFATc2 interface 75 3.4 Conclusion 77 Chapter 4 - Exploring UBLs & UBL-Interaction Motifs: Computational & 78 Experimental analysis of ubiquilin, NFATc2IP, UIMs and SIMs. 4.1 Introduction 79 4.1.1 Database & comparative analysis 79 4.1.1.1 Similarities & differences between model family members 80 4.1.1.2 Common defining features for each modelling family 80 4.2 Experimental Procedures 81 4.2.1 UBL Database Development 81 4.2.2 Relating 17 structurally determined UBLs to nearest neighbours 82 and model families 4.2.3 Secondary structure prediction & analysis 83 4.2.4 Relating structural features to functional pathways 83 4.3 Results 84 4.3.1 Structurally characterized ubiquitin-like domains 84 4.3.2 Nearest-neighbours of ubiquitin-like domains 85 4.3.3 Nearest-neighbours of structurally characterized UBMs 86 4.3.4 Grouping UBLs based on biological processes and 89 molecular function viii Table of Contents (continued) 4.3.5 Grouping UBLs based on medical significance 91 4.3.5.1 Cellular localization 92 4.3.6 Grouping UBLs based on cell localization 93 4.4 Conclusion 95 Chapter 5 - Conclusion and Future Directions 96 5.1 Conclusions 96 5.2 Future Directions 97 5.2.1 Ubiquitin-like domain fold, NFATc2IP & ubiquilins 97 5.2.2 Ubiquitin-like domain structural genomics 98 5.2.3 Protein Domain family analyses 98 5.3 Concluding remarks 98 Chapter 6 - References 99 ix List of Tables Table 1.1: List of 18 annotated ubiquitin-like modifiers, and associated enzymatic 8 complement, substrates and functional pathways. Table 1.2: Protein-protein interaction modes structurally characterized with 14 experimentally determined binding affinities between UBLs and binding partners. Table 2.1: Summary of small-scale expression screening of human ubiquitin-like 29 domains structurally characterized and deposited in the PDB as part of this thesis. Table 2.2: Summary of 1H15N-HSQC screening results for human ubiquitin-like 30 domains. 10 ubiquitin-like domains were solved by NMR (red), and 7 ubiquitin-like domains were solved by X-ray crystallography (blue).
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