Abstract
The E3 ligase parkin ubiquitinates outer mitochondrial membrane proteins during oxidative stress and is linked to early‐onset Parkinson's disease. Parkin is autoinhibited but is activated by the kinase PINK1 that phosphorylates ubiquitin leading to parkin recruitment, and stimulates phosphorylation of parkin's N‐terminal ubiquitin‐like (pUbl) domain. How these events alter the structure of parkin to allow recruitment of an E2~Ub conjugate and enhanced ubiquitination is an unresolved question. We present a model of an E2~Ub conjugate bound to the phospho‐ubiquitin‐loaded C‐terminus of parkin, derived from NMR chemical shift perturbation experiments. We show the UbcH7~Ub conjugate binds in the open state whereby conjugated ubiquitin binds to the RING1/IBR interface. Further, NMR and mass spectrometry experiments indicate the RING0/RING2 interface is re‐modelled, remote from the E2 binding site, and this alters the reactivity of the RING2(Rcat) catalytic cysteine, needed for ubiquitin transfer. Our experiments provide evidence that parkin phosphorylation and E2~Ub recruitment act synergistically to enhance a weak interaction of the pUbl domain with the RING0 domain and rearrange the location of the RING2(Rcat) domain to drive parkin activity.
Highlights
Parkinson’s disease is the second most common neurodegenerative disease estimated to affect 1% of the population over 60 years of age (Tysnes & Storstein, 2017)
We show that E2-Ub recruitment to parkin results in two distinct types of NMR chemical shift changes: one set that is consistent with the E2~Ub binding site and a second set that corresponds to residues near the RING0/RING2 interface, indicating this region is re-modelled during E2~Ub recruitment
Structures of near full-length parkin (Kumar et al, 2015; Sauveet al, 2015) show the C-terminal RING0–RING1–IBR–RING2(Rcat) domains form a compact unit whereby the N-terminal Ubl domain interacts with the RING1 and IBR domains and portions of the tether region (Fig 1A). This mode of interaction confirmed that the Ubl domain exerted its previously identified autoinhibitory effect (Chaugule et al, 2011) by blocking the expected binding site on the RING1 domain from an E2 conjugating enzyme resulting in negligible ubiquitination activity
Summary
Parkinson’s disease is the second most common neurodegenerative disease estimated to affect 1% of the population over 60 years of age (Tysnes & Storstein, 2017). In addition to sporadic Parkinson’s disease, there are genetic forms of the disease that account for approximately 10% of all cases. Mutations in the genes for PARK2 and PARK6 give rise to earlyonset or autosomal recessive juvenile parkinsonism (ARJP) forms of the disease that have similar symptoms including rigidity, bradykinesia and postural instability (Jankovic, 2008) but affect individuals at a much younger age. PARK2 encodes the E3 ubiquitin ligase parkin (Kitada et al, 1998) where mutations account for 50% of all ARJP cases. Along with the PTEN-induced kinase (PINK1) translated from PARK6, these proteins use the ubiquitin degradation pathway to turnover damaged mitochondria and maintain mitochondrial homeostasis, especially under conditions of oxidative stress
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