Cryo-EM structure of gastric H+,K+-ATPase with a single occupied cation-binding site

Gastric H+, K+-ATPase is responsible for gastric acid secretion. ATP-driven H+ uptake into the stomach is efficiently accomplished by the exchange of an equal amount of K+, resulting in a luminal pH close to 1. Because of the limited free energy available for ATP hydrolysis, the stoichiometry of transported cations is thought to vary from 2H(+)/2K(+) to 1H(+)/1K(+) per hydrolysis of one ATP molecule as the luminal pH decreases, although direct evidence for this hypothesis has remained elusive. Here, we show, using the phosphate analog aluminum fluoride (AlF) and a K+ congener (Rb+), the 8-angstrom resolution structure of H+, K+-ATPase in the transition state of dephosphorylation, (Rb+)E2 similar to AlF, which is distinct from the preceding Rb+-free E2P state. A strong density located in the transmembrane cation-binding site of (Rb+) E2 similar to AlF highly likely represents a single bound Rb+ ion, which is clearly different from the Rb+-free E2AlF or K+-bound (K+) E2 similar to AlF structures. Measurement of radioactive Rb-86(+) binding suggests that the binding stoichiometry varies depending on the pH, and approximately half of the amount of Rb+ is bound under acidic crystallization conditions compared with at a neutral pH. These data represent structural and biochemical evidence for the 1H(+)/1K(+)/1ATP transport mode of H+, K+-ATPase, which is a prerequisite for generation of the 10(6)-fold proton gradient in terms of thermodynamics. Together with the released E2P-stabilizing interaction between the beta subunit's N terminus and the P domain observed in the (Rb+) E2 similar to AlF structure, we propose a refined vectorial transport model of H+, K+-ATPase, which must prevail against the highly acidic state of the gastric lumen.