Cryo-EM and MD infer water-mediated proton transport and autoinhibition mechanisms of Vo complex

Abstract

Rotary vacuolar adenosine triphosphatases (V-ATPases) drive transmembrane proton transport through a Vo proton channel subcomplex. Despite recent high-resolution structures of several rotary ATPases, the dynamic mechanism of proton pumping remains elusive. Here, we determined a 2.7-angstrom cryo-electron microscopy (cryo-EM) structure of yeast V-o proton channel in nanodisc that reveals the location of ordered water molecules along the proton path, details of specific protein-lipid interactions, and the architecture of the membrane scaffold protein. Moreover, we uncover a state of V-o that shows the c-ring rotated by similar to 14 degrees. Molecular dynamics simulations demonstrate that the two rotary states are in thermal equilibrium and depict how the protonation state of essential glutamic acid residues couples water-mediated proton transfer with c-ring rotation. Our cryo-EM models and simulations also rationalize a mechanism for inhibition of passive proton transport as observed for free V-o that is generated as a result of V-ATPase regulation by reversible disassembly in vivo.