biostudies-literatureAbe KMEXT | Japan Society for the Promotion of ScienceNaito FoundationTakeda Science FoundationJapan Agency for Medical Research and DevelopmentOno Medical Research Foundation5709https://www.ebi.ac.uk/biostudies/studies/S-EPMC8481561biostudies-literatureUnknown12(1)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.Nature communicationsGastric proton pump with two occluded K⁺ engineered with sodium pump-mimetic mutations.PMC8481561JP19am010107421H02426Yamamoto KOshima AIrie KAbe KNishizawa TfalseGastric 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.2021-01-01T00:00:00Z2021 Sep2026-06-15T04:51:38.793Z2025-02-18T23:52:45.064ZS-EPMC84815613458845310.1038/s41467-021-26024-1