Doctor of Philosophy
Shaw, Gary S.
The E2-ubiquitin conjugate is a key regulator of ubiquitination and is therefore an important component of cellular homeostasis. Disruptions to proper E2-ubiquitin functioning have implications in diseases such as shigellosis and Parkinson’s disease discussed here. E2-ubiquitin conjugates like UbcH7-ubiquitin are extremely dynamic and can adopt multiple conformations in solution or bound to target proteins. However, the conformational arrangements that UbcH7-ubiquitin adopts while free in solution, bound to the shigellosis-associated kinase OspG or to the Parkinson’s disease-related E3 ligase parkin are unknown. Also unknown, is a mechanistic explanation for how UbcH7-ubiquitin interactions with OspG and parkin are associated with disease. Here, we determined the crystal structure of OspG bound to UbcH7-ubiquitin, the crystal structure of autoinhibited full-length human parkin with and without a phosphorylation-mimetic, the crystal and NMR structures of activated full-length human parkin bound to a phosphorylated-ubiquitin molecule, and an NMR structure of activated human parkin bound to both phosphorylated-ubiquitin and UbcH7-ubiquitin. This work determined that UbcH7-ubiquitin predominantly occupies closed states in solution but binds to OspG and parkin in open conformations. Further key findings include showing that UbcH7-ubiquitin is a biological target of OspG and that OspG involvement in shigellosis is to halt host ubiquitination by competitively binding to UbcH7-ubiquitin in a way that mimics host HECT E3 binding. We showed that parkin is autoinhibited through interdomain interactions. Phosphorylation of autoinhibited parkin primes phosphorylated-ubiquitin binding and this binding relieves autoinhibition by inducing allosteric rearrangements in parkin to allow subsequent UbcH7-ubiquitin engagement. Finally, we showed that certain hereditary variants in parkin are likely associated with autosomal recessive juvenile parkinsonism due to a loss in the ability to interact with UbcH7-ubiquitin. Research here has significant implications for understanding the basis of shigellosis and hereditary forms of Parkinson’s disease, and has contributed significant molecular understandings for the use in developing therapeutics.
Condos, Tara E. C., "Conformational Arrangements of UbcH7-Ubiquitin with OspG and Parkin" (2018). Electronic Thesis and Dissertation Repository. 5330.
Available for download on Thursday, April 30, 2020