7ET1

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

(2021) Nat Commun 12: 5709-5709

• PubMed: 34588453 Search on PubMedSearch on PubMed Central
• DOI: https://doi.org/10.1038/s41467-021-26024-1
• Primary Citation of Related Structures:  
  7EFL, 7EFM, 7EFN, 7ET1


• PubMed 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.

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Organizational Affiliation: 

Cellular and Structural Physiology Institute, Nagoya University, Nagoya, 464-8601, Japan. [email protected].