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

Zev. A Ripstein, Rui Huang, Rafal Augustyniak, Lewis E. Kay, John L. Rubinstein
doi: https://doi.org/10.1101/105866
Zev. A Ripstein
1The Hospital for Sick Children Research Institute, Toronto, ON, Canada M5G 0A4
2Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8
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Rui Huang
2Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8
3Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8
4Department of Chemistry, University of Toronto, Toronto, ON, Canada M5S 3H6
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Rafal Augustyniak
2Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8
3Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8
4Department of Chemistry, University of Toronto, Toronto, ON, Canada M5S 3H6
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Lewis E. Kay
1The Hospital for Sick Children Research Institute, Toronto, ON, Canada M5G 0A4
2Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8
3Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8
4Department of Chemistry, University of Toronto, Toronto, ON, Canada M5S 3H6
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  • For correspondence: kay@pound.med.utoronto.ca john.rubinstein@utoronto.ca
John L. Rubinstein
1The Hospital for Sick Children Research Institute, Toronto, ON, Canada M5G 0A4
2Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8
5Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada M5G 1L7
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  • For correspondence: kay@pound.med.utoronto.ca john.rubinstein@utoronto.ca
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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.

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Posted February 04, 2017.
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Structure of a AAA+ unfoldase in the process of unfolding substrate
Zev. A Ripstein, Rui Huang, Rafal Augustyniak, Lewis E. Kay, John L. Rubinstein
bioRxiv 105866; doi: https://doi.org/10.1101/105866
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Structure of a AAA+ unfoldase in the process of unfolding substrate
Zev. A Ripstein, Rui Huang, Rafal Augustyniak, Lewis E. Kay, John L. Rubinstein
bioRxiv 105866; doi: https://doi.org/10.1101/105866

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