Abstract
The Ser/Thr protein kinase PINK1 phosphorylates the well‐folded, globular protein ubiquitin (Ub) at a relatively protected site, Ser65. We previously showed that Ser65 phosphorylation results in a conformational change in which Ub adopts a dynamic equilibrium between the known, common Ub conformation and a distinct, second conformation wherein the last β‐strand is retracted to extend the Ser65 loop and shorten the C‐terminal tail. We show using chemical exchange saturation transfer (CEST) nuclear magnetic resonance experiments that a similar, C‐terminally retracted (Ub‐CR) conformation also exists at low population in wild‐type Ub. Point mutations in the moving β5 and neighbouring β‐strands shift the Ub/Ub‐CR equilibrium. This enabled functional studies of the two states, and we show that while the Ub‐CR conformation is defective for conjugation, it demonstrates improved binding to PINK1 through its extended Ser65 loop, and is a superior PINK1 substrate. Together our data suggest that PINK1 utilises a lowly populated yet more suitable Ub‐CR conformation of Ub for efficient phosphorylation. Our findings could be relevant for many kinases that phosphorylate residues in folded protein domains.
Highlights
Protein ubiquitination and protein phosphorylation are the two main regulatory post-translational modifications of proteins (Hunter, 2007)
The pathophysiological importance of PINK1/Parkin-mediated mitophagy is underlined by the fact that mutations in PINK1 and Parkin are linked to autosomal recessive juvenile Parkinson’s disease (ARJP), a neurodegenerative condition arising from loss of dopaminergic neurons in the substantia nigra (Corti et al, 2011; Pickrell & Youle, 2015)
Dynamic aspects of Ub have been under intense scrutiny, in particular by nuclear magnetic resonance (NMR), and numerous studies have collectively covered most motional timescales from fast ps internal motions up to ls-ms conformational exchange processes using RDC analysis (Lange et al, 2008; Torchia, 2015)
Summary
Protein ubiquitination and protein phosphorylation are the two main regulatory post-translational modifications of proteins (Hunter, 2007). Ub phosphorylation at Ser has been linked to mitophagy, the process by which damaged parts of mitochondria are isolated and targeted for autophagic clearance (Pickrell & Youle, 2015; Nguyen et al, 2016). PINK1 phosphorylates Ub attached to outer mitochondrial membrane proteins, and this recruits and allosterically activates the E3 ligase Parkin (Kazlauskaite et al, 2015; Kumar et al, 2015; Sauveet al, 2015; Wauer et al, 2015b). PINK1 phosphorylates Parkin in its Ub-like (Ubl) domain, which is required for full Parkin activation and leads to strong, localised mitochondrial ubiquitination (Kondapalli et al, 2012; Ordureau et al, 2014; Wauer et al, 2015b). The pathophysiological importance of PINK1/Parkin-mediated mitophagy is underlined by the fact that mutations in PINK1 and Parkin are linked to autosomal recessive juvenile Parkinson’s disease (ARJP), a neurodegenerative condition arising from loss of dopaminergic neurons in the substantia nigra (Corti et al, 2011; Pickrell & Youle, 2015)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
