Conformational dynamics and asymmetry in multimodal inhibition of membrane-bound pyrophosphatases

Membrane-bound pyrophosphatases (mPPases) are homodimeric proteins that hydrolyse pyrophosphate and pump H+/Na+ across membranes. They are crucial for the virulence of protist pathogens, making them attractive drug targets. In this study, we investigate the inhibitory effects of seven distinct bisphosphonates against Thermotoga maritima mPPase to explore their mode of action and assist in future small molecule inhibitor development. We solved two structures of mPPase bound to the inhibitors in the enzyme active sites and probed the conformational dynamics of mPPase under multiple inhibitors and functionally relevant conditions by double electron-electron resonance (DEER) spectroscopy. We found that mPPase adopts five distinct conformations within the ensemble in the presence of different inhibitors. Combined with solid-supported membrane-based electrophysiology recordings, this revealed that during catalysis, one monomer of the dimer remains open, and Na+ can only be pumped in a closed state. These results further support the existence of catalytic asymmetry in our pumping-before-hydrolysis model.