Abstract
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.
Highlights
The gastric H+,K+-ATPase mediates electroneutral exchange of 1H+/1K+ per ATP hydrolysed across the membrane
Coupled ATP hydrolysis and ion transport is achieved by large structural changes throughout the enzyme and these have been well studied at the molecular level, especially for SERCA and NKA, through numerous crystal structures and functional analyses[11,12,13]
In the presence of K+, beryllium fluoride (BeF), and -competitive acid blocker (P-CAB), the enzyme is trapped in the P-CAB·K+·E2P state via the reverse reaction. b Overall structure of HKA Lys791Ser/ Glu820Asp/Tyr340Asn/Glu936Val quadruple mutant in BYK99 and Rb+-bound E2BeF state determined in this study, is shown in ribbon representations
Summary
The gastric H+,K+-ATPase mediates electroneutral exchange of 1H+/1K+ per ATP hydrolysed across the membrane. The cation-binding site is formed in the middle of the membrane domain, surrounded by TM4, TM5, TM6, and TM8 in both HKA and NKA (Fig. 1).
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