Gastric proton pump with two occluded K+ engineered with sodium pump-mimetic mutations

The gastric H+,K+-ATPase mediates electroneutral exchange of 1H+/1K+ per ATP hydrolysed across the membrane. Previous structural analysis of the K+-occluded E2-Pi form of H+,K+-ATPase showed a single bound K+ at cation-binding site II, in marked contrast to the two K+ occluded at sites I and II of the closely-related Na+,K+-ATPase which mediates electrogenic 3Na+/2K+ translocation across the membrane. The two pumps show significant differences in structure in and around Site I, but which are critical for blocking K+ binding in the gastric pump and contribute to binding in the sodium pump is unclear. We have a series of crystal structures and a cryo-EM structure of H+,K+-ATPase mutants with changes in the vicinity of site I based on the structure of the sodium pump. The number of bound Rb+, determined by its anomalous dispersion, remains one in the luminal-open E2BeF form of the Lys791Ser single mutant and Lys791Ser/Glu820Asp double mutant, mutation that could create space and may directly bind the cation. We next introduced mutations in peripheral residues Try340Asn and Glu936Val. A strong and spread-out Rb+ anomalous density observed in the quadruple mutant suggests that a certain population ATPases has two Rb+ bound. We then added gate-closing mutation Try799Trp and determined its cryo-EM structure in the occluded E2-AlF form. This quintuple mutant unambiguously has two separate densities at the cation-binding site. The step-wise construction of the K+ binding site offers new insight into how it is blocked in the one pump and constituted in the other.