An invisible ubiquitin conformation is required for efficient phosphorylation by PINK1

Christina Gladkova, Alexander F. Schubert, Jane L. Wagstaff, Jonathan N. Pruneda, Stefan M.V. Freund, David Komander

Research output: Contribution to journalArticlepeer-review

38 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)3555-3572
Number of pages18
JournalEMBO Journal
Issue number24
StatePublished - Dec 15 2017
Externally publishedYes


  • PINK1
  • Parkin
  • Parkinson's disease
  • nuclear magnetic resonance
  • ubiquitin phosphorylation

ASJC Scopus subject areas

  • Neuroscience(all)
  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)


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