PT  - JOURNAL ARTICLE
AU  - Christina Gladkova
AU  - Alexander F. Schubert
AU  - Jane L. Wagstaff
AU  - Jonathan N. Pruneda
AU  - Stefan M.V. Freund
AU  - David Komander
TI  - An ‘invisible’ ubiquitin conformation is required for efficient phosphorylation by PINK1
AID  - 10.1101/189027
DP  - 2017 Jan 01
TA  - bioRxiv
PG  - 189027
4099  - http://biorxiv.org/content/early/2017/09/21/189027.short
4100  - http://biorxiv.org/content/early/2017/09/21/189027.full
AB  - The Ser/Thr protein kinase PINK1 phosphorylates the well-folded, globular protein ubiquitin (Ub) at a relatively protected site, Ser65. We had previously shown 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 in which the last β-strand is retracted to extend the Ser65 loop and shorten the C-terminal tail. We here show using Chemical Exchange Saturation Transfer (CEST) NMR experiments, that a similar, C-terminally retracted (Ub-CR) conformation exists in wild-type Ub. Ub point mutations in the moving β5-strand and in neighbouring strands shift the Ub/Ub-CR equilibrium. This enabled functional studies of the two states, and we show that the Ub-CR conformation binds to the PINK1 kinase domain 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 proteins or domains.