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| Title | An unusual conformation from Na-sensitive non-gastric proton pump mutants reveals molecular mechanisms of cooperative Na-binding. |
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| Journal, issue, pages | Biochim Biophys Acta Mol Cell Res, Vol. 1870, Issue 7, Page 119543, Year 2023 |
| Publish date | Jul 22, 2023 |
 Authors | Kazuhiro Abe / Tomohiro Nishizawa / Pablo Artigas /   |
| PubMed Abstract | The 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 ...The 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 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. |
 External links | Biochim Biophys Acta Mol Cell Res / PubMed:37482134 |
| Methods | EM (single particle) |
| Resolution | 2.62 - 3.65 Å |
| Structure data | EMDB-35488, PDB-8ijl: EMDB-35489, PDB-8ijm:  EMDB-35491: Cryo-EM structure of non-gastric proton pump K794S mutant in Na+ bound E1AMPPCP state |
| Chemicals |  ChemComp-ALF:  ChemComp-MG:  ChemComp-ADP:  ChemComp-PCW:  ChemComp-CLR:  ChemComp-NA:  ChemComp-Q7G:  ChemComp-NAG:  ChemComp-HOH:  ChemComp-ACP: |
| Source |
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 Keywords |  MEMBRANE PROTEIN / P-type ATPase / P2-type ATPase |
