Structure of the Lipid Nanodisc-reconstituted Vacuolar ATPase Proton Channel

Abstract

Eukaryotic vacuolar H+-ATPase (V-ATPase) is a multi-subunit 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 electron microscopy (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. Three-dimensional (3-D) reconstructions calculated at ~15 A 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 (ITC) 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 enzyme's central rotor. Comparison of the three V-ATPase conformations to 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.