title: Models for the a subunits of the Thermus thermophilus V/A-ATPase and Saccharomyces cerevisiae V-ATPase enzymes by cryo-EM and evolutionary covariance.
abstract: Rotary ATPases couple ATP synthesis or hydrolysis to proton translocation across a membrane. However, understanding proton translocation has been hampered by a lack of structural information for the membrane-embedded a subunit. The V/A-ATPase from the eubacterium Thermus thermophilus is similar in structure to the eukaryotic V-ATPase but has a simpler subunit composition and functions in vivo to synthesize ATP rather than pump protons. We determined the T. thermophilus V/A-ATPase structure by cryo-EM at 6.4 Å resolution. Evolutionary covariance analysis allowed tracing of the a subunit sequence within the map, providing a complete model of the rotary ATPase. Comparing the membrane-embedded regions of the T. thermophilus V/A-ATPase and eukaryotic V-ATPase from Saccharomyces cerevisiae allowed identification of the α-helices that belong to the a subunit and revealed the existence of previously unknown subunits in the eukaryotic enzyme. Subsequent evolutionary covariance analysis enabled construction of a model of the a subunit in the S. cerevisae V-ATPase that explains numerous biochemical studies of that enzyme. Comparing the two a subunit structures determined here with a structure of the distantly related a subunit from the bovine F-type ATP synthase revealed a conserved pattern of residues, suggesting a common mechanism for proton transport in all rotary ATPases.
authors.affiliation: The Hospital for Sick Children Research Institute, Toronto, ON, Canada M5G0A4; Department of Medical Biophysics, The University of Toronto, Toronto, ON, Canada M5G1L7;
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Schep
authors.affiliation: The Hospital for Sick Children Research Institute, Toronto, ON, Canada M5G0A4; Department of Medical Biophysics, The University of Toronto, Toronto, ON, Canada M5G1L7;
authors.forename: Jianhua
authors.identifier: http://orcid.org/0000-0002-4997-766X
authors.initials: J
authors.lastname: Zhao
authors.affiliation: The Hospital for Sick Children Research Institute, Toronto, ON, Canada M5G0A4; Department of Medical Biophysics, The University of Toronto, Toronto, ON, Canada M5G1L7; Department of Biochemistry, The University of Toronto, Toronto, ON, Canada M5S1A8 john.rubinstein@utoronto.ca.
authors.forename: John L
authors.initials: JL
authors.lastname: Rubinstein
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D005075:Biological Evolution; D020285:Cryoelectron Microscopy / Q000379:methods*; D008954:Models, Biological; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D016963:Thermus thermophilus / Q000201:enzymology*
chemical_list: D000251:Adenosine Triphosphatases
keywords: V-ATPase; V/A-ATPase; cryo-EM; evolutionary covariance; structure