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

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 (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.Ph.D