Abstract
Vacuolar H(+)-ATPases (V-ATPases; V(1)V(o)-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 (V(1)ΔH). 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 V(1)ΔH with high affinity. We also found that only one molecule of H(NT) binds V(1)ΔH with high affinity, suggesting conformational asymmetry of the three EG heterodimers in V(1)ΔH. Moreover, MgATP hydrolysis-driven conformational changes in V(1) destabilized the interaction of H or H(NT) with V(1)ΔH, 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.