Date of Award

2021

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Biochemistry & Molecular Biophysics

First Advisor

A. Joshua Wand

Abstract

Parkin is an E3 ligase, a ubiquitin writer that ligates single or chains of ubiquitin onto its substrates, thereby modulating their cellular levels, localization, and function in downstream signaling pathways. Together with upstream E1 and E2 ligases, downstream deubiquitinases and ubiquitin-interacting proteins, Parkin regulates a variety of target proteins. Parkin activity has been shown to induce mitophagy of damaged mitochondria and prevent apoptotic cell death. Owing to its role in mitochondrial regulation, Parkin protects against neurodegeneration, cardiac disease, lung damage, inflammation, and cancer, becoming a therapeutic target of interest.

Parkin is allosterically regulated, with auto-inhibited Parkin being recruited to the surface of damaged mitochondria and activated by two events - binding of phosphorylated ubiquitin and phosphorylation of its N-terminal auto-inhibitory domain. These activating interactions have been shown to facilitate large allosteric conformational changes in the Parkin monomer exposing the formerly buried catalytic site. Our knowledge of these conformational changes comes from structural and biochemical studies of truncated Parkin constructs. However, it is yet unclear what mechanisms drive Parkin's allosteric regulation or how its auto-inhibitory and activating interactions influence each other in the context of the full-length protein. Studying these mechanisms is critical to understanding Parkin function and modifying it for therapeutic purposes.

The goal of this dissertation is to establish the foundations to study allostery in Parkin by high-resolution NMR spectroscopy and other biophysical methods. This dissertation reports the first ever expression and purification of full-length human Parkin in biophysical quantities, under optimized conditions for NMR sample stability. Backbone chemical shifts of Parkin have been assigned using standard triple resonance experiments and selective unlabelling strategies. Despite technical challenges posed by large protein size and difficult sample conditions, in-house automated assignment methods have yielded confident assignments in all domains of Parkin, including the inter-domain linkers that are visualized for the first time. This dissertation also provides insights into local secondary structure based on backbone assignments and discusses preliminary work on studying Parkin stability by the complementary method of Native-hydrogen exchange. This dissertation sets the stage to studying Parkin solution dynamics and energetics, and their role in its allosteric regulation.

Embargoed

Available to all on Friday, January 31, 2025

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