Structure of a AAA+ unfoldase in the process of unfolding substrate
- The Hospital for Sick Children Research Institute, Canada
- University of Toronto, Canada
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
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VCP like ATPase from T. acidophilum (VAT) - Conformation 1Publicly available at the EMBL-EMD Protein Data Bank in Europe (accession no. EMD-8658).
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VCP like ATPase from T. acidophilum (VAT) - Substrate bound conformationPublicly available at the EMBL-EMD Protein Data Bank in Europe (accession no. EMD-8659.
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VCP like ATPase from T. acidophilum (VAT) - Conformation 1Publicly available at the RCSB Protein Data Bank (accession no. 5VC7.
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VCP like ATPase from T. acidophilum (VAT) - Substrate bound conformationPublicly available at the RCSB Protein Data Bank (accession no. 5VCA).
Article and author information
Author details
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
© 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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Structure of a AAA+ unfoldase in the process of unfolding substrate
eLife 6:e25754.
https://doi.org/10.7554/eLife.25754
Further reading
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In healthy cells, cyclin D1 is expressed during the G1 phase of the cell cycle, where it activates CDK4 and CDK6. Its dysregulation is a well-established oncogenic driver in numerous human cancers. The cancer-related function of cyclin D1 has been primarily studied by focusing on the phosphorylation of the retinoblastoma (RB) gene product. Here, using an integrative approach combining bioinformatic analyses and biochemical experiments, we show that GTSE1 (G-Two and S phases expressed protein 1), a protein positively regulating cell cycle progression, is a previously unrecognized substrate of cyclin D1–CDK4/6 in tumor cells overexpressing cyclin D1 during G1 and subsequent phases. The phosphorylation of GTSE1 mediated by cyclin D1–CDK4/6 inhibits GTSE1 degradation, leading to high levels of GTSE1 across all cell cycle phases. Functionally, the phosphorylation of GTSE1 promotes cellular proliferation and is associated with poor prognosis within a pan-cancer cohort. Our findings provide insights into cyclin D1’s role in cell cycle control and oncogenesis beyond RB phosphorylation.
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