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-P transition state of H + ,K + -ATPase showed a single bound K + at cation-binding site II, in marked contrast to the two K + ions occluded at sites I and II of the closely-related Na + ,K + -ATPase which mediates electrogenic 3Na + /2K + translocation across the membrane. The molecular basis of the different K + stoichiometry between these K + -counter-transporting pumps is elusive. We show 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. Our step-wise and tailored construction of the mutants finally gave a two-K + bound H + ,K + -ATPase, achieved by five mutations, including amino acids directly coordinating K + (Lys791Ser, Glu820Asp), indirectly contributing to cation-binding site formation (Tyr340Asn, Glu936Val), and allosterically stabilizing K + -occluded conformation (Tyr799Trp). This quintuple mutant in the K + -occluded E2-P state unambiguously shows two separate densities at the cation-binding site in its 2.6 Å resolution cryo-EM structure. These results offer new insights into how two closely-related cation pumps specify the number of K + accommodated at their cation-binding site. The gastric H + ,K + -ATPase is a proton pump that creates the acidic environment of the stomach lumen, maintaining high proton gradient across the gastric mucosa cell membrane. Here, structural analysis of rationally designed H + ,K + -ATPase mutants provides insight into this and other P-type ATPases cation binding stoichiometry and mechanisms.