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From Proteopedia
Cyo-EM structure of K794A non-gastric proton pump in Na+ bound E1AMPPCP state
Structural highlights
FunctionAT12A_RAT The catalytic subunit of a H(+)/K(+) ATPase and/or Na(+)/K(+) ATPase pump which transports K(+) ions in exchange for Na(+) and/or H(+) ions across the apical membrane of epithelial cells. Uses ATP as an energy source to pump K(+) ions into the cell while transporting Na(+) and/or H(+) ions to the extracellular compartment (PubMed:10644526, PubMed:7560093). Involved in the maintenance of electrolyte homeostasis through K(+) ion absorption in kidney and colon (By similarity). In the airway epithelium, may play a primary role in mucus acidification regulating its viscosity and clearance (By similarity).[UniProtKB:P54707][UniProtKB:Q9Z1W8][1] [2] Publication Abstract from PubMedThe Na(+),K(+)-ATPase (NKA) and non-gastric H(+),K(+)- ATPase (ngHKA) share ~65 % sequence identity, and nearly identical catalytic cycles. These pumps alternate between inward-facing (E1) and outward-facing (E2) conformations and differ in their exported substrate (Na(+) or H(+)) and stoichiometries (3 Na(+):2 K(+) or 1 H(+):1 K(+)). We reported that structures of the NKA-mimetic ngHKA mutant K794S/A797P/W940/R949C (SPWC) with 2 K(+) occluded in E2-P(i) and 3 Na(+)-bound in E1.ATP states were nearly identical to NKA structures in equivalent states. Here we report the cryo-EM structures of K794A and K794S, two poorly-selective ngHKA mutants, under conditions to stabilize the E1.ATP state. Unexpectedly, the structures show a hybrid with both E1- and E2-like structural features. While transmembrane segments TM1-TM3 and TM4's extracellular half adopted an E2-like conformation, the rest of the protein assumed an E1 configuration. Two spherical densities, likely bound Na(+), were observed at cation-binding sites I and III, without density at site II. This explains the E2-like conformation of TM4's exoplasmic half. In NKA, oxygen atoms derived from the unwound portion of TM4 coordinated Na(+) at site II. Thus, the lack of Na(+) at site II of K794A/S prevents the luminal portion of TM4 from taking an E1-like position. The K794A structure also suggests that incomplete coordination of Na(+) at site III induces the halfway rotation of TM6, which impairs Na(+)-binding at the site II. Thus, our observations provide insight into the molecular mechanism of E2-E1 transition and cooperative Na(+)-binding in the NKA and other related cation pumps. An unusual conformation from Na(+)-sensitive non-gastric proton pump mutants reveals molecular mechanisms of cooperative Na(+)-binding.,Abe K, Nishizawa T, Artigas P Biochim Biophys Acta Mol Cell Res. 2023 Oct;1870(7):119543. doi: , 10.1016/j.bbamcr.2023.119543. Epub 2023 Jul 22. PMID:37482134[3] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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