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

Abstract
Data availability
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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
1. Rubinstein et al.
(2017) VCP like ATPase from T. acidophilum (VAT) - Conformation 1
Publicly available at the RCSB Protein Data Bank (accession no. 5VC7.

http://www.rcsb.org/pdb/explore/explore.do?structureId=5VC7
1. Rubinstein et al.
(2017) VCP like ATPase from T. acidophilum (VAT) - Substrate bound conformation
Publicly available at the RCSB Protein Data Bank (accession no. 5VCA).

http://www.rcsb.org/pdb/explore/explore.do?structureId=5VCA

Article and author information

Author details

Zev A Ripstein

The Hospital for Sick Children Research Institute, Toronto, Canada

Competing interests
No competing interests declared.
Rui Huang

Department of Biochemistry, University of Toronto, Toronto, Canada

Competing interests
No competing interests declared.
Rafal Augustyniak

Department of Biochemistry, University of Toronto, Toronto, Canada

Competing interests
No competing interests declared.
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.

"This ORCID iD identifies the author of this article:" 0000-0002-4054-4083
John L Rubinstein

The Hospital for Sick Children Research Institute, Toronto, Canada

For correspondence
john.rubinstein@utoronto.ca

Competing interests
No competing interests declared.

"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.

Copyright

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.