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Title: The mechanism of Parkin activation by PINK1 phosphorylation
Author: Gladkova, Christina
ISNI:       0000 0004 7968 6315
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
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Mutations in the E3 Ub ligase Parkin (PARK2) and the protein kinase PINK1 (PARK6) are genetically linked to Young-Onset Parkinson's Disease. The two enzymes orchestrate clearance of damaged, cytotoxic mitochondria through a specialized form of autophagy termed mitophagy. By phosphorylating ubiquitin (Ub) on the surface of damaged mitochondria, PINK1 generates a phospho-Ub receptor for autoinhibited cytosolic Parkin. Once localized to the mitochondrial surface, Parkin is activated by direct PINK1-mediated phosphorylation on its Ub-like domain (Ubl). To efficiently recruit the autophagy machinery, activated Parkin transfers Ub from upstream E2 Ub-conjugating enzymes to substrates on the mitochondrial surface via an E3~Ub intermediate. Resolving the precise mechanism of Parkin activation by PINK1-mediated phosphorylation is the primary aim of this thesis. Additionally, building on previous work in which a novel phospho-Ub conformer was characterized crystallographically, the same conformer was found in unmodified Ub in this work. This striking finding is supported by further biochemical and biophysical investigation of the newly discovered equilibrium in unmodified Ub. To address the mechanism of Parkin activation and open new avenues for translational research, both dynamic and crystallographic approaches were employed. Hydrogen-deuterium exchange mass spectrometry reveals that Parkin phosphorylation enables a conformational equilibrium between an autoinhibited and an active Parkin state. In the active state the phospho-Ubl domain binds to the Unique Parkin domain (UPD) of the Parkin core and thereby displaces the catalytic RING2 domain from its autoinhibitory position. Catalytic intermediates, such as the E2-bound state or the Ub-charged Parkin species shift the equilibrium in favour of the active conformer. Parkin-mediated substrate ubiquitination occurs by a flexibly tethered catalytic RING2 domain independently of the Parkin core, in line with its minimal substrate selectivity observed in vitro. The new activating interface between the Parkin phospho-Ubl and the Parkin core is revealed in a 1.8Å crystal structure of phosphorylated human Parkin lacking the flexible catalytic domain. Additionally to the phospho-Ubl, a conserved linker region, the activating element (ACT) aids displacement of the catalytic RING2 domain by mimicking RING2 autoinhibitory interactions with the Parkin core. This crystal structure explains patient mutations in the UPD phosphate-binding pocket as well as in the newly identified ACT. Together, the clinically relevant molecular insights described in this thesis may facilitate the development of therapeutic or diagnostic tools for Parkinson's disease.
Supervisor: Komander, David Sponsor: Medical Research Council (MRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Parkin ; Ubiquitin ; Parkinson's disease ; E3 ligase ; Mitophagy ; phosphorylation ; nuclear magnetic resonance ; hydrogen-deuterium exchange mass spectrometry ; structural biology