Solution Structure Of Human Small Ubiquitin-related Modifier SUMO-3 C47S And Its Interaction With SUMO Conjugating Enzyme Ubc9

NMR is a useful tool to solve solution structure of proteins and to study their interactions with other molecules. Combined with computational biology, NMR can provide more detailed information on elucidating how proteins play their biological roles in vivo at atomic level. Our work focuses on the cloning, expression, purification and structural studies of SUMO-3 C47S, a truncated protein with C47S mutation of the small ubiquitin-related modifier SUMO-3. The solution structure of SUMO-3 C47S has been determined by NMR spectroscopy. Furthermore, we investigate its interaction with SUMO conjugating enzyme Ubc9.Chapter 1 provides a brief review of SUMO modification, including its discovery, biochemical reaction process and biological functions in vivo. SUMO was primarily identified in Saccharomyces cerevisiae. Subsequent studies show that SUMO, about 100 amino acids, is ubiquitiously expressed and highly conserved in all eukaryotes. Post-translational modification with SUMO, termed sumoylation, has emerged as an important cellular regulatory mechanism. SUMO functions by covalently and reversibly modifying other proteins. SUMO modification of cellular proteins is a multistep cascade reaction which many enzymes are involved in. Similar to ubiquitination, this pathway requires the activating enzyme El and conjugating enzyme E2, and in most cases, requires a ligating enzyme E3. Including SUMO and a substrate protein, the modification pathway involves five proteins and requires several reactions that lead to the conjugation between SUMO and a substrate protein. The linkage between them is an isopeptide bond between the C-terminal glycine of SUMO and the ε-amino group of a lysine residue in the substrate. This lysine is frequently found at a conserved ΨKXE motif, where Ψ is a hydrophobic amino acid residue and X is any residue. During the conjugation process, various non-covalent and covalent complexes are formed transiently among these proteins. Unlike ubiquitin which mostly tags proteins for degradation, SUMO has many important cellular functions, such as nucleocytoplasmic trafficking, protein localization, transcription regulation, signal transduction, antagonizing ubiquitination, cell cycle and genome integrity. The present studies suggest that SUMO family members are both overlapped and distinct in cellular localization and functions.In chapter 2, the solution structure of SUMO-3 C47S was determined by NMR. We also study its interaction with SUMO conjugating enzyme Ubc9 using docking approach in combination with NMR chemical shift mapping information. Recombinant SUMO-3 C47S was cloned and expressed in E.coli. SUMO-3 C47S was purified using Ni-chelating column and then sephadex G-50 gel filtration. The solution structure of SUMO-3 C47S, which was obtained by heteronuclear three-dimensional spectroscopy, was similar to other SUMO family members featuring the β-β-α-β-β-α-β ubiquitin fold. A di-glycine motif at the C-terminal tail, extending away from the core structure, is accessible to enzymes and substrates. Despite the similar protein fold, the surface charge distributions of SUMO are very...