Phosphorylation by PINK1 releases the UBL domain and initializes the conformational opening of the E3 ubiquitin ligase Parkin.

Thomas R Caulfield,Wolfdieter Springer,Samuel C Flores,Daniel F A R Dourado,Elisabeth L Moussaud-Lamodière,Fabienne C Fiesel,Sergei L Kosakovsky Pond

doi:10.1371/journal.pcbi.1003935

Thomas R Caulfield, Wolfdieter Springer + Show 5 more

Open Access

https://doi.org/10.1371/journal.pcbi.1003935

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Abstract

Loss-of-function mutations in PINK1 or PARKIN are the most common causes of autosomal recessive Parkinson's disease. Both gene products, the Ser/Thr kinase PINK1 and the E3 Ubiquitin ligase Parkin, functionally cooperate in a mitochondrial quality control pathway. Upon stress, PINK1 activates Parkin and enables its translocation to and ubiquitination of damaged mitochondria to facilitate their clearance from the cell. Though PINK1-dependent phosphorylation of Ser65 is an important initial step, the molecular mechanisms underlying the activation of Parkin's enzymatic functions remain unclear. Using molecular modeling, we generated a complete structural model of human Parkin at all atom resolution. At steady state, the Ub ligase is maintained inactive in a closed, auto-inhibited conformation that results from intra-molecular interactions. Evidently, Parkin has to undergo major structural rearrangements in order to unleash its catalytic activity. As a spark, we have modeled PINK1-dependent Ser65 phosphorylation in silico and provide the first molecular dynamics simulation of Parkin conformations along a sequential unfolding pathway that could release its intertwined domains and enable its catalytic activity. We combined free (unbiased) molecular dynamics simulation, Monte Carlo algorithms, and minimal-biasing methods with cell-based high content imaging and biochemical assays. Phosphorylation of Ser65 results in widening of a newly defined cleft and dissociation of the regulatory N-terminal UBL domain. This motion propagates through further opening conformations that allow binding of an Ub-loaded E2 co-enzyme. Subsequent spatial reorientation of the catalytic centers of both enzymes might facilitate the transfer of the Ub moiety to charge Parkin. Our structure-function study provides the basis to elucidate regulatory mechanisms and activity of the neuroprotective Parkin. This may open up new avenues for the development of small molecule Parkin activators through targeted drug design.

Highlights

Mutations in the PTEN-induced putative kinase 1 (PINK1) and PARKIN genes are the most common causes of autosomal recessive Parkinson’s disease (PD) [1]
The molecular mechanism underlying the pathogenesis of PD remain elusive, it has become clear that PINK1 and Parkin protein functionally cooperate in a novel mitochondrial quality control pathway [2]
The linker is comprised of two sub-domains: (1) a semi-globular domain from residues 77 to that appears highly dynamic and (2) a tethering loop region from residues to 140 that connects to the RING0 domain

Summary

Introduction

Mutations in the PTEN-induced putative kinase 1 (PINK1) and PARKIN genes are the most common causes of autosomal recessive Parkinson’s disease (PD) [1]. Adaptor proteins such as p97 and p62 are recruited to facilitate clustering of mitochondria around perinuclear regions and the selective degradation of substrates via the proteasome system and of whole organelles via autophagy (mitophagy) [3,7,9,10]. Mutations in both genes, PINK1 and PARKIN, abrogate this presumably neuroprotective pathway through distinct molecular mechanisms and at different steps along the sequential process [3,4,5,6]

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