title: Structure of a AAA+ unfoldase in the process of unfolding substrate.
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, theThermoplasma acidophilumhomologue 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.
authors.affiliation: The Hospital for Sick Children Research Institute, Toronto, Canada.|Department of Biochemistry, University of Toronto, Toronto, Canada.
authors.forename: Zev A
authors.identifier: 0000-0003-3601-0596
authors.initials: ZA
authors.lastname: Ripstein
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, Canada.|Department of Molecular Genetics, University of Toronto, Toronto, Canada.|Department of Chemistry, University of Toronto, Toronto, Canada.
authors.forename: Rui
authors.initials: R
authors.lastname: Huang
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, Canada.|Department of Molecular Genetics, University of Toronto, Toronto, Canada.|Department of Chemistry, University of Toronto, Toronto, Canada.
authors.forename: Rafal
authors.initials: R
authors.lastname: Augustyniak
authors.affiliation: The Hospital for Sick Children Research Institute, Toronto, Canada.|Department of Biochemistry, University of Toronto, Toronto, Canada.|Department of Molecular Genetics, University of Toronto, Toronto, Canada.|Department of Chemistry, University of Toronto, Toronto, Canada.
authors.forename: Lewis E
authors.identifier: 0000-0002-4054-4083
authors.initials: LE
authors.lastname: Kay
authors.affiliation: The Hospital for Sick Children Research Institute, Toronto, Canada.|Department of Biochemistry, University of Toronto, Toronto, Canada.|Department of Medical Biophysics, University of Toronto, Toronto, Canada.
authors.forename: John L
authors.identifier: 0000-0003-0566-2209
authors.initials: JL
authors.lastname: Rubinstein
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D019843:Archaeal Proteins / Q000378:metabolism* / Q000648:ultrastructure*; D020285:Cryoelectron Microscopy; D008958:Models, Molecular; D011487:Protein Conformation; D013822:Thermoplasma / Q000201:enzymology*; D000074405:Valosin Containing Protein / Q000378:metabolism* / Q000648:ultrastructure*
chemical_list: D019843:Archaeal Proteins; D000255:Adenosine Triphosphate; D000074405:Valosin Containing Protein; C105134:vat protein, Thermoplasma acidophilum
keywords: AAA+; ATPase; Cryo-EM; Thermoplasma acidophilum; VAT; biochemistry; biophysics; structural biology; unfolding