MgATP hydrolysis destabilizes the interaction between subunit H and yeast V1-ATPase, highlighting H's role in V-ATPase regulation by reversible disassembly

Vacuolar H+-ATPases (V-ATPases; V1Vo-ATPases) are rotary-motor proton pumps that acidify intracellular compartments and, in some tissues, the extracellular space. V-ATPase is regulated by reversible disassembly into autoinhibited V-1-ATPase and V-o proton channel sectors. An important player in V-ATPase regulation is subunit H, which binds at the interface of V-1 and V-o. H is required for MgATPase activity in holo-V-ATPase but also for stabilizing the MgADP-inhibited state in membrane-detached V-1. However, how H fulfills these two functions is poorly understood. To characterize the H-V-1 interaction and its role in reversible disassembly, we determined binding affinities of full-length H and its N-terminal domain (H-NT) for an isolated heterodimer of subunits E and G (EG), the N-terminal domain of subunit a (a(NT)), and V-1 lacking subunit H (V1H). Using isothermal titration calorimetry (ITC) and biolayer interferometry (BLI), we show that H-NT binds EG with moderate affinity, that full-length H binds a(NT) weakly, and that both H and H-NT bind V1H with high affinity. We also found that only one molecule of H-NT binds V1H with high affinity, suggesting conformational asymmetry of the three EG heterodimers in V1H. Moreover, MgATP hydrolysis-driven conformational changes in V-1 destabilized the interaction of H or H-NT with V1H, suggesting an interplay between MgADP inhibition and subunit H. Our observation that H binding is affected by MgATP hydrolysis in V-1 points to H's role in the mechanism of reversible disassembly.