title: Structure of the Lipid Nanodisc-reconstituted Vacuolar ATPase Proton Channel: DEFINITION OF THE INTERACTION OF ROTOR AND STATOR AND IMPLICATIONS FOR ENZYME REGULATION BY REVERSIBLE DISSOCIATION.
abstract: Eukaryotic vacuolar H+-ATPase (V-ATPase) is a multisubunit enzyme complex that acidifies subcellular organelles and the extracellular space. V-ATPase consists of soluble V1-ATPase and membrane-integral Voproton channel sectors. To investigate the mechanism of V-ATPase regulation by reversible disassembly, we recently determined a cryo-EM reconstruction of yeast VoThe structure indicated that, when V1is released from Vo, the N-terminal cytoplasmic domain of subunit a (aNT) changes conformation to bind rotor subunit d However, insufficient resolution precluded a precise definition of the aNT-d interface. Here we reconstituted Vointo lipid nanodiscs for single-particle EM. 3D reconstructions calculated at ∼15-Å resolution revealed two sites of contact between aNTand d that are mediated by highly conserved charged residues. Alanine mutagenesis of some of these residues disrupted the aNT-d interaction, as shown by isothermal titration calorimetry and gel filtration of recombinant subunits. A recent cryo-EM study of holo V-ATPase revealed three major conformations corresponding to three rotational states of the central rotor of the enzyme. Comparison of the three V-ATPase conformations with the structure of nanodisc-bound Vorevealed that Vois halted in rotational state 3. Combined with our prior work that showed autoinhibited V1-ATPase to be arrested in state 2, we propose a model in which the conformational mismatch between free V1and Vofunctions to prevent unintended reassembly of holo V-ATPase when activity is not needed.
authors.affiliation: From the Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Stam
authors.affiliation: From the Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210. Electronic address: wilkenss@upstate.edu.
authors.forename: Stephan
authors.initials: S
authors.lastname: Wilkens
mesh_terms: D008563:Membrane Lipids / Q000737:chemistry*; D049329:Nanostructures / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry*
chemical_list: D008563:Membrane Lipids; D029701:Saccharomyces cerevisiae Proteins; D025262:Vacuolar Proton-Translocating ATPases
keywords: EM; Vo proton channel; isothermal titration calorimetry (ITC); lipid nanodisc; protein structure; proton transport; vacuolar ATPase