1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics

Structure of a AAA+ unfoldase in the process of unfolding substrate

  1. Zev A Ripstein
  2. Rui Huang
  3. Rafal Augustyniak
  4. Lewis E Kay  Is a corresponding author
  5. John L Rubinstein  Is a corresponding author
  1. The Hospital for Sick Children Research Institute, Canada
  2. University of Toronto, Canada
https://doi.org/10.7554/eLife.25754
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  1. Zev A Ripstein
  2. Rui Huang
  3. Rafal Augustyniak
  4. Lewis E Kay
  5. John L Rubinstein
(2017)
Structure of a AAA+ unfoldase in the process of unfolding substrate
eLife 6:e25754.
https://doi.org/10.7554/eLife.25754
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Abstract

AAA+ unfoldases are thought to unfold substrate through the central pore of their hexameric structures, but how this process occurs is not known. VAT, the Thermoplasma acidophilum homologue of eukaryotic CDC48/p97, works in conjunction with the proteasome to degrade misfolded or damaged proteins. We show that in the presence of ATP, VAT with its regulatory N-terminal domains removed unfolds other VAT complexes as substrate. We captured images of this transient process by electron cryomicroscopy (cryo-EM) to reveal the structure of the substrate-bound intermediate. Substrate binding breaks the six-fold symmetry of the complex, allowing five of the six VAT subunits to constrict into a tight helix that grips an ~80 Å stretch of unfolded protein. The structure suggests a processive hand-over-hand unfolding mechanism, where each VAT subunit releases the substrate in turn before re-engaging further along the target protein, thereby unfolding it.

Data availability

The following data sets were generated
    1. Rubinstein et al.
    (2017) VCP like ATPase from T. acidophilum (VAT) - Conformation 1
    Publicly available at the EMBL-EMD Protein Data Bank in Europe (accession no. EMD-8658).
    http://www.ebi.ac.uk/pdbe/entry/emdb/EMD-8658
    1. Rubinstein et al.
    (2017) VCP like ATPase from T. acidophilum (VAT) - Substrate bound conformation
    Publicly available at the EMBL-EMD Protein Data Bank in Europe (accession no. EMD-8659.
    http://www.ebi.ac.uk/pdbe/entry/emdb/EMD-8659

Article and author information

Author details

  1. Zev A Ripstein

    The Hospital for Sick Children Research Institute, Toronto, Canada
    Competing interests
    No competing interests declared.

  2. Rui Huang

    Department of Biochemistry, University of Toronto, Toronto, Canada
    Competing interests
    No competing interests declared.

  3. Rafal Augustyniak

    Department of Biochemistry, University of Toronto, Toronto, Canada
    Competing interests
    No competing interests declared.

  4. Lewis E Kay

    The Hospital for Sick Children Research Institute, Toronto, Canada
    For correspondence
    kay@pound.med.utoronto.ca
    Competing interests
    Lewis E Kay, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4054-4083

  5. John L Rubinstein

    The Hospital for Sick Children Research Institute, Toronto, Canada
    For correspondence
    john.rubinstein@utoronto.ca
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0566-2209

Funding

Canadian Institutes of Health Research (MOP133408 MOP81294)

  • John L Rubinstein

Natural Sciences and Engineering Research Council of Canada

  • Zev A Ripstein

Canada Research Chairs

  • John L Rubinstein

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Edward H Egelman, University of Virginia, United States

Publication history

  1. Received: February 6, 2017
  2. Accepted: April 6, 2017
  3. Accepted Manuscript published: April 8, 2017 (version 1)
  4. Version of Record published: May 9, 2017 (version 2)

Copyright

© 2017, Ripstein et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Zev A Ripstein
  2. Rui Huang
  3. Rafal Augustyniak
  4. Lewis E Kay
  5. John L Rubinstein
(2017)
Structure of a AAA+ unfoldase in the process of unfolding substrate
eLife 6:e25754.
https://doi.org/10.7554/eLife.25754

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