Doctor of Philosophy
Dr. Gary S. Shaw
Mutations in the gene encoding parkin, an E3-ubiquitin ligase, result in 50% of Autosomal Recessive Juvenile Parkinsonism cases. Parkin has been identified as a key mediator of mitochondrial regeneration following oxidative stress, and pathogenic mutations have been shown to impair its ubiquitin ligase activity. Neurodegeneration of dopamine-producing neurons appears to be a downstream consequence of parkin loss-of-function, resulting in early-onset forms of Parkinson’s disease. Although ubiquitination activity is essential for its neuroprotective function, parkin is autoinhibited in its native state by various mechanisms, including its N-terminal ubiquitin-like (UBL) domain. Therefore, the overarching objective of this thesis was to structurally characterize the autoinhibited state of parkin and determine how this inactive structure is modified to a catalytically-competent form.
It was determined that autoinhibited parkin maintains a compact tertiary structure mediated by a tight intramolecular association between the UBL and C-terminal region. A high-resolution NMR strategy was developed and used to identify the binding site of the UBL domain that further revealed allosteric structural changes associated with UBL binding and displacement.
Recently, multiple reports emerged identifying serine 65 phosphorylation of both ubiquitin and parkin’s UBL domain as key inducers of parkin activity. To examine the roles of these phosphorylation signals, several methods were used to generate homogenous S65-phosphorylated ubiquitin and UBL including: chemical modification, orthogonal translation, and phosphorylation by a catalytically-active kinase. Thermodynamic parameters of ubiquitin and UBL binding to parkin were measured and it was demonstrated how these are significantly altered upon S65 phosphorylation to promote a loss of autoinhibition.
To understand the structural consequences of S65 phosphorylation, the three-dimensional structure of the phosphorylated parkin UBL was solved. Phosphorylation impacts the structure, stability and autoinhibitory association of the UBL domain in parkin. Further, cooperative roles of phospho-ubiquitin and phospho-UBL were established in reorganizing the parkin to an extended structure, allowing its engagement in the ubiquitination cascade.
Finally, to investigate a chemical mechanism of catalysis in activated parkin, a detailed characterization of active site atoms in parkin was performed. Chemical biology approaches were used to generate an activated parkin~ubiquitin intermediate that will provide further mechanistic insight into ubiquitin transfer onto mitochondrial substrates.
Aguirre, Jacob D., "Autoinhibition and Activation of Parkin" (2017). Electronic Thesis and Dissertation Repository. 4516.
Available for download on Friday, March 01, 2019