PINK1 autophosphorylation is required for ubiquitin recognition
Shafqat Rasool1,2, Naoto Soya3,#, Luc Truong1,#, Nathalie Croteau1, Gergely L Lukacs2,3 & Jean-François Trempe1,*
1Department of Pharmacology & Therapeutics and Groupe de Recherche Axé sur la Structure des Protéines, McGill University, Montréal, QC, H3G 1Y6, Canada2Department of Biochemistry, McGill University, Montréal, QC, H3G 1Y6, Canada3Department of Physiology and Groupe de Recherche Axé sur la Structure des Protéines, McGill University, Montréal, QC, H3G 1Y6, Canada
Mutations in PINK1 cause autosomal recessive Parkinson's disease (PD), a neurodegenerative movement disorder. PINK1 is a kinase that acts as a sensor of mitochondrial damage and initiates Parkin-mediated clearance of the damaged organelle. PINK1 phosphorylates Ser65 in both ubiquitin and the ubiquitin-like (Ubl) domain of Parkin, which switch on its E3 ligase activity. Autophosphorylation of PINK1 is required for Parkin activation, but how this modulates the ubiquitin kinase activity is unclear. Here, we show that autophosphorylation of Tribolium castaneum PINK1 is required for substrate recognition. Using enzyme kinetics and NMR spectroscopy, we reveal that PINK1 binds the Parkin Ubl with a 10-fold higher affinity than ubiquitin via a conserved interface that is also implicated in RING1 and SH3 binding. The interaction requires phosphorylation at Ser205, an invariant PINK1 residue (Ser228 in human). Using mass spectrometry, we demonstrate that PINK1 rapidly autophosphorylates in trans at Ser205. Hydrogen-deuterium exchange experiments provide insights into the structure of the PINK1 catalytic domain, and reveal how Ser205 phosphorylation affects the dynamics of the canonical C-helix and activation loop in the active site. Our findings suggest that multiple PINK1 molecules autophosphorylate first prior to binding and phosphorylating ubiquitin and Parkin.