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.

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 Vo proton channel sectors. To investigate the mechanism of V-ATPase regulation by reversible disassembly, we recently determined a cryo-EM reconstruction of yeast Vo The structure indicated that, when V1 is 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 Vo into lipid nanodiscs for single-particle EM. 3D reconstructions calculated at ∼15-Å resolution revealed two sites of contact between aNT and 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 Vo revealed that Vo is 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 V1 and Vo functions to prevent unintended reassembly of holo V-ATPase when activity is not needed.