title: Unfolding the mechanism of the AAA+ unfoldase VAT by a combined cryo-EM, solution NMR study.
abstract: The AAA+ (ATPases associated with a variety of cellular activities) enzymes play critical roles in a variety of homeostatic processes in all kingdoms of life. Valosin-containing protein-like ATPase of Thermoplasma acidophilum (VAT), the archaeal homolog of the ubiquitous AAA+ protein Cdc48/p97, functions in concert with the 20S proteasome by unfolding substrates and passing them on for degradation. Here, we present electron cryomicroscopy (cryo-EM) maps showing that VAT undergoes large conformational rearrangements during its ATP hydrolysis cycle that differ dramatically from the conformational states observed for Cdc48/p97. We validate key features of the model with biochemical and solution methyl-transverse relaxation optimized spectroscopY (TROSY) NMR experiments and suggest a mechanism for coupling the energy of nucleotide hydrolysis to substrate unfolding. These findings illustrate the unique complementarity between cryo-EM and solution NMR for studies of molecular machines, showing that the structural properties of VAT, as well as the population distributions of conformers, are similar in the frozen specimens used for cryo-EM and in the solution phase where NMR spectra are recorded.
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8; Department of Chemistry, University of Toronto, Toronto, ON, Canada M5S 3H6; Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8;
authors.forename: Rui
authors.initials: R
authors.lastname: Huang
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8; The Hospital for Sick Children Research Institute, Toronto, ON, Canada M5G 0A4;
authors.forename: Zev A
authors.initials: ZA
authors.lastname: Ripstein
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8; Department of Chemistry, University of Toronto, Toronto, ON, Canada M5S 3H6; Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8;
authors.forename: Rafal
authors.initials: R
authors.lastname: Augustyniak
authors.affiliation: Laboratory of Bioinformatics and Systems Biology, Centre of New Technologies, University of Warsaw, 02-089 Warsaw, Poland; Department of Physical Chemistry, Faculty of Pharmacy, Medical University of Warsaw, 02-097 Warsaw, Poland;
authors.forename: Michal
authors.initials: M
authors.lastname: Lazniewski
authors.affiliation: Laboratory of Bioinformatics and Systems Biology, Centre of New Technologies, University of Warsaw, 02-089 Warsaw, Poland;
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Ginalski
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8; Department of Chemistry, University of Toronto, Toronto, ON, Canada M5S 3H6; Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8; The Hospital for Sick Children Research Institute, Toronto, ON, Canada M5G 0A4; kay@pound.med.utoronto.ca john.rubinstein@utoronto.ca.
authors.forename: Lewis E
authors.initials: LE
authors.lastname: Kay
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8; The Hospital for Sick Children Research Institute, Toronto, ON, Canada M5G 0A4; Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada M5G 1L7 kay@pound.med.utoronto.ca john.rubinstein@utoronto.ca.
authors.forename: John L
authors.initials: JL
authors.lastname: Rubinstein
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics; D020285:Cryoelectron Microscopy; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D013822:Thermoplasma / Q000201:enzymology / Q000235:genetics; D000074405:Valosin Containing Protein / Q000737:chemistry* / Q000235:genetics
chemical_list: D019843:Archaeal Proteins; D000074405:Valosin Containing Protein; C105134:vat protein, Thermoplasma acidophilum
keywords: VAT; cryo-EM; methyl-TROSY NMR; proteasome; protein dynamics