Identification of Glu779 as an Essential Part of the Cation Binding Site of the Sodium Pump

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-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.

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

Nature Communications ◽

10.1038/s41467-021-26024-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Kazuhiro Abe ◽

Kenta Yamamoto ◽

Katsumasa Irie ◽

Tomohiro Nishizawa ◽

Atsunori Oshima

Keyword(s):

Amino Acids ◽

Structural Analysis ◽

Binding Site ◽

Molecular Basis ◽

Sodium Pump ◽

Cation Binding ◽

Site Formation ◽

Marked Contrast ◽

Cation Binding Site ◽

Gastric Proton Pump

AbstractThe 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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