Structure of the Lipid Nanodisc-reconstituted Vacuolar ATPase Proton Channel

Eukaryotic vacuolar H+-ATPase (V-ATPase) is a multisub-unit enzyme complex that acidifies subcellular organelles and the extracellular space. V-ATPase consists of soluble V-1-ATPase and membrane-integral V-o proton channel sectors. To investigate the mechanism of V-ATPase regulation by reversible disassembly, we recently determined a cryo-EM reconstruction of yeast V-o. The structure indicated that, when V-1 is released from V-o, the N-terminal cytoplasmic domain of subunit a (a(NT)) changes conformation to bind rotor subunit d. However, insufficient resolution precluded a precise definition of the a(NT)-d interface. Here we reconstituted V-o into lipid nanodiscs for single-particle EM. 3D reconstructions calculated at similar to 15-angstrom resolution revealed two sites of contact between a(NT) and d that are mediated by highly conserved charged residues. Alanine mutagenesis of some of these residues disrupted the a(NT)-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 V-o revealed that V-o is halted in rotational state 3. Combined with our prior work that showed autoinhibited V-1-ATPase to be arrested in state 2, we propose a model in which the conformational mismatch between free V-1 and V-o functions to prevent unintended reassembly of holo V-ATPase when activity is not needed.