Article | Published:

Structure of PINK1 in complex with its substrate ubiquitin

Nature volume 552, pages 5156 (07 December 2017) | Download Citation

Abstract

Autosomal-recessive juvenile Parkinsonism (AR-JP) is caused by mutations in a number of PARK genes, in particular the genes encoding the E3 ubiquitin ligase Parkin (PARK2, also known as PRKN) and its upstream protein kinase PINK1 (also known as PARK6). PINK1 phosphorylates both ubiquitin and the ubiquitin-like domain of Parkin on structurally protected Ser65 residues, triggering mitophagy. Here we report a crystal structure of a nanobody-stabilized complex containing Pediculus humanus corporis (Ph)PINK1 bound to ubiquitin in the ‘C-terminally retracted’ (Ub-CR) conformation. The structure reveals many peculiarities of PINK1, including the architecture of the C-terminal region, and reveals how the N lobe of PINK1 binds ubiquitin via a unique insertion. The flexible Ser65 loop in the Ub-CR conformation contacts the activation segment, facilitating placement of Ser65 in a phosphate-accepting position. The structure also explains how autophosphorylation in the N lobe stabilizes structurally and functionally important insertions, and reveals the molecular basis of AR-JP-causing mutations, some of which disrupt ubiquitin binding.

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Acknowledgements

We thank beamline scientists at Diamond Light Source (DLS) for support at beamlines I04-1, I02 and I24; INSTRUCT, part of the European Strategy Forum on Research Infrastructures (ESFRI) and the Research Foundation - Flanders (FWO) for support with Nanobody discovery; A. Lundqvist for technical assistance during nanobody discovery; P. R. Elliott for help with crystallography and data collection; M. A. Michel, J. N. Pruneda, M. Gersch and other members of the D.K. laboratory for advice, reagents and discussions; and D. Barford and R. Williams for discussions and advice. Access to DLS was supported in part by the EU FP7 infrastructure grant BIOSTRUCT-X (contract 283570). The D.K. laboratory is supported by the Medical Research Council (U105192732), the European Research Council (309756, 724804), the Michael J. Fox Foundation and the Lister Institute for Preventive Medicine.

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Affiliations

  1. Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK

    • Alexander F. Schubert
    • , Christina Gladkova
    • , Jane L. Wagstaff
    • , Stefan M. V. Freund
    • , Sarah L. Maslen
    •  & David Komander
  2. VIB-VUB Center for Structural Biology, VIB, 1050 Brussels, Belgium.

    • Els Pardon
    •  & Jan Steyaert
  3. Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussels, Belgium

    • Els Pardon
    •  & Jan Steyaert

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Contributions

A.F.S. and D.K. designed the study, and A.F.S. performed all experiments. C.G., S.M.V.F. and J.L.W. designed and characterized Ub-CR reagents, S.L.M. performed mass spectrometry, and E.P. and J.S. generated nanobodies. D.K. wrote the manuscript.

Competing interests

D.K. is part of the DUB Alliance, which includes Cancer Research Technology and FORMA Therapeutics.

Corresponding author

Correspondence to David Komander.

Reviewer Information Nature thanks J. W. Harper, T. Hunter and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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    Supplementary Information

    This file contains Supplementary Figure 1, the uncropped versions of all SDS-PAGE gels and Supplementary Table 1, collated AR-JP mutations and their predicted structural effects.

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DOI

https://doi.org/10.1038/nature24645

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