An ‘invisible’ ubiquitin conformation is required for efficient phosphorylation by PINK1

Abstract

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.

Analysis of the Ub to Ub-CR Transition in Ubiquitin

“…The retraction can therefore occur while Leu67 and Ala4 are still in contact, resulting in a lower barrier and a smaller number of backbone rearrangements after the retraction. The F4A mutation was introduced to shift the equilibrium towards UbCR, which is indeed observed in experiment, 13 and consistent with the change in our predicted mechanism due to the lack of stabilisation of Ub by Phe4-Leu67…”

“…The overall structure consists of two funnels, one for Ub and one for Ub-CR, as expected from the two-state behaviour observed in experiment. 13 enthalpic factors stabilising this structure, leading to an interesting transition in the free energy landscapes and the pathways between Ub and Ub-CR.…”

“…[5][6][7][8] Phosphorylation at Ser65 is achieved by the kinase PINK1, [9][10][11][12] but until recently this process was poorly understood, as the canonical ubiquitin fold (Ub) does not expose Ser65, and furthermore, the residue forms hydrogen bonds with Gln62. 13 Recently it was shown that ubiquitin exhibits another conformation, 13 which had previously only been observed for phosphorylated ubiquitin. 12,14 In the newly described conformation (Ub-CR) the C-terminal tail is retracted, exposing Ser65.…”

PINK 1 autophosphorylation is required for ubiquitin recognition

“…We observed a more widespread broadening in the titration with 2 H, 15 N‐labeled Ub (Fig EV2C). We attribute this primarily to relaxation broadening from residual 1 H in Ub, combined with the slower tumbling induced by oligomerization that takes place in TcPINK1 at high concentrations (required to saturate the weaker binding), or to chemical exchange broadening arising from widespread structural changes in Ub in the PINK1‐bound form [; see Discussion]. Nevertheless, we could unambiguously identify the amides of residues 46–49 in Ub as undergoing the fastest signal loss upon TcPINK1 binding (Fig C), which also show selective broadening in the Ubl.…”

“…Thus, Ub may be binding to a larger complex, where residual non‐exchangeable 1 H, as well as exchangeable 1 H, contribute further to 1 H‐ 1 H dipole‐induced T2 relaxation, independently of the proximity to TcPINK1. Alternatively, the group of David Komander observed that Ub is adopting an alternative conformation with a 2 a.a. shift in the last β strand . This low‐population conformation is proposed to be the form that binds to PINK1 for efficient Ser65 phosphorylation.…”