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Structure insights into the autoinhibitory mechanism of the deubiquitinating enzyme USP25 and into the SUMO E1-E2 protein-protein recognition Bing Liu Supervisor: Dr. David Reverter A thesis submitted for the degree of Biochemistry and Molecular Biology Universitat Autònoma de Barcelona Institut de Biotecnologia i de Biomedicina (IBB) September 2018 The pursuit of science needs special courage Acknowledgements First of all, I would like to thank my mother and sisters for their love and support at every moment, despite a distance of ten thousand kilometers away. To my mother, I know you have been worried about my lives in Barcelona. I apologize that I cannot take care of you these years, albeit you never complain about that. To my two sisters, I thank you for the kindly help on my lives. I also would like to thank my girlfriend for your support when I was feeling frustrated and helpless and for the wonderful time we had together. To my supervisor, David Reverter, I would like to sincerely thank you for your best training on my scientific research. Your knowledge and ideas of science always inspire me, and your patient guide let me have a glimpse into the beauty of protein structure at the first time. I would like to thank my lab mates, Natalia, Jara, Pablo, Zhen, as well as members from our neighbor group, for your kindly help and discussion on my research. I am also thankful to members of the other groups in IBB, for the help on the lab work. I would like to thank Maria Lois from CRAG-UAB, Marta Sureda-Gómez and Virginia Amador from IDIBAPS, for their direct contributions on my research publications. Finally, thanks to Chinese Scholarship Council (CSC) grant for supporting my PhD study. Bing Liu September 2018 Index Introduction………...……………………………………………....….1 The ubiquitin system and deubiquitinating enzyme USP25….……….….….3 The SUMO system and E1-E2 protein-protein recognition…...………...….17 USP25 in the cross talk of ubiquitin and SUMO pathways……………..…..23 Research objectives………..…………….……………………………..…..35 Chapter 1: A quaternary tetramer assembly inhibits the deubiquitinating activity of USP25…………………………...41 Chapter 2: Structural analysis and evolution of specificity of the SUMO UFD E1-E2 interactions………………………93 Chapter 3: Structure insights into specificities of SUMO E1-E2 interactions between A. thaliana and human………………125 General conclusions ………………...…………………………….………165 General discussion……………………………..………………….….…...171 Summary in English…………………..………………………..…………..181 Summary in Catalán…………………..……...…………………………….185 Introduction 1 2 The ubiquitin system and deubiquitinating enzyme USP25 Post-translational modifications increase protein complexity While it is estimated that human genome encodes 20,000-25,000 protein- coding genes (1), the total number of proteins in human proteome is estimated to be many times more, possibly ranging from 10,000 to almost 1 million. This is because a single gene can generate different variants of a particular protein through, for example, alternative splicing, mRNA editing, and genomic recombination. The complexity of human proteome is further increased by protein post-translational modifications (PTMs), including phosphorylation, glycosylation, lipidation, carbonylation, acetylation, methylation, and ubiquitination. These mechanisms can somehow explain why humans could be so much more complex with just 25,000 genes. Figure 1. A view of post-translational modifications from protein complexity (2). Ubiquitin and ubiquitination In the late 1970s and 1980s, Avram Hershko, Aaron Ciechanover and Irwin Rose discovered and characterized the ATP-dependent, ubiquitin-mediated proteolysis. As a result of their pioneering studies, they were awarded with the 2004 Nobel Prize in Chemistry (3). After the landmark discovery of ubiquitin-mediated degradation by the 26S proteasome, the protein-based modifications became 3 prevalent. Today, it is quite clear that this function of ubiquitin was just the tip of an enormous iceberg. The recent global proteomic studies provide insight into the complexity of the ubiquitin system and about 1.3% of total cellular proteins are substrates of ubiquitination in human cells (4). In human societies, people communicate with each other using words and sentences that can trigger specific responses from other individuals. Similarly, proteins are modified with polymeric ubiquitin chains, in which the linkage between ubiquitin molecules encodes information. Thus, ubiquitin code is like the communication language between and within cells and determinates the fate of its substrate. The ubiquitin system is a universal means of protein regulation which controls a wide range of cellular processes including gene transcription, cell cycle, cell death, signal transduction, DNA repair, and autophagy (5-10). Worth of the name, ubiquitin is quite ubiquitous in biology. Ubiquitin (Ub) is a highly conserved 76-residue protein (~8.5 kDa) that is present in all eukaryotes, from yeast to human. In human genome, there are four genes coding for ubiquitin: UBB, UBC, UBA52, and RPS27A (11). Ubiquitin-like proteins (Ubls) are a set of proteins which adopt the same β-grasp fold as ubiquitin. As one of the most known protein posttranslational modifiers, ubiquitin and Ubls are covalently attached to an ε-amino group of the substrate protein’s lysine residues (Figure 2). This modification is carried out via a conserved three-step enzymatic cascade through the E1 activating enzyme, the E2 conjugation enzyme, and E3 protein ligase, resulting the formation of an isopeptide bond between the C terminus of ubiquitin and the lysine on the target protein (5, 12). First, E1 activates the C-terminal glycine residue of ubiquitin in an ATP-dependent manner, resulting in the formation of an intermediate ubiquitin adenylate, followed by the binding of the C-terminus of ubiquitin to the active cysteine residue of E1 through a thioester linkage. Second, the activated ubiquitin is transferred from E1 to E2, whose active cysteine residue forms a thioester bond with the C-terminus of ubiquitin. Finally, E3 ligase interacts with ubiquitin-loaded E2 and mediates the final ubiquitin transfer, forming an isopeptide bond between the lysine ε-amino group of the target protein and the C-terminal carboxyl group of ubiquitin. In addition, ubiquitin can also be attached on N-terminus amino group, cysteine, serine, and threonine residues of the target proteins (13-17). 4 Figure 2. Ubl conjugation and deconjugation pathways (10). Precursor Ubls (including ubiquitin) are processed by either DUBs (deubiquitinating enzymes) or ULPs (Ubl-specific proteases) to expose a C-terminal glycine in the mature Ubl. The processed Ubl is conjugated to the target protein via a three-step enzymatic cascade through the E1 activating enzyme, E2 conjugation enzyme, and E3 protein ligase. The DUBs and ULPs can remove Ubls from substrates. Ubiquitination enzymes Specificity of ubiquitination to the thousands of human substrates depends on the sequential action of E1s, E2s, and E3s. There are 2 E1s, ~40 E2s, and more than 600 E3s in human ubiquitin system (18-23). E2s play the dominant role in the ubiquitin chain assembly. E3s are the most heterogeneous and critical components in the ubiquitination cascade, as they bind directly to their target proteins and strictly control the efficiency and specificity of the ubiquitination reaction (21).