An unusual conformation from Na+-sensitive non-gastric proton pump mutants reveals molecular mechanisms of cooperative Na+-binding
Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved..
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-Pi 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.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2023 |
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| Erschienen: |
2023 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:1870 |
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| Enthalten in: |
Biochimica et biophysica acta. Molecular cell research - 1870(2023), 7 vom: 01. Okt., Seite 119543 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Abe, Kazuhiro [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 16.08.2023 Date Revised 16.08.2023 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1016/j.bbamcr.2023.119543 |
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| Förderinstitution / Projekttitel: |
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| 100 | 1 | |a Abe, Kazuhiro |e verfasserin |4 aut | |
| 245 | 1 | 3 | |a An unusual conformation from Na+-sensitive non-gastric proton pump mutants reveals molecular mechanisms of cooperative Na+-binding |
| 264 | 1 | |c 2023 | |
| 336 | |a Text |b txt |2 rdacontent | ||
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| 500 | |a Date Completed 16.08.2023 | ||
| 500 | |a Date Revised 16.08.2023 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved. | ||
| 520 | |a 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-Pi 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, U.S. Gov't, Non-P.H.S. | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 4 | |a Colonic proton pump | |
| 650 | 4 | |a Cryo-EM | |
| 650 | 4 | |a Membrane proteins | |
| 650 | 4 | |a Non-gastric proton pump | |
| 650 | 4 | |a P-type ATPases | |
| 650 | 4 | |a Sodium pump | |
| 650 | 7 | |a Proton Pumps |2 NLM | |
| 650 | 7 | |a Sodium |2 NLM | |
| 650 | 7 | |a 9NEZ333N27 |2 NLM | |
| 650 | 7 | |a Adenosine Triphosphate |2 NLM | |
| 650 | 7 | |a 8L70Q75FXE |2 NLM | |
| 650 | 7 | |a Adenosine Triphosphatases |2 NLM | |
| 650 | 7 | |a EC 3.6.1.- |2 NLM | |
| 700 | 1 | |a Nishizawa, Tomohiro |e verfasserin |4 aut | |
| 700 | 1 | |a Artigas, Pablo |e verfasserin |4 aut | |
| 773 | 0 | 8 | |i Enthalten in |t Biochimica et biophysica acta. Molecular cell research |d 2017 |g 1870(2023), 7 vom: 01. Okt., Seite 119543 |w (DE-627)NLM264216326 |x 1879-2596 |7 nnns |
| 773 | 1 | 8 | |g volume:1870 |g year:2023 |g number:7 |g day:01 |g month:10 |g pages:119543 |
| 856 | 4 | 0 | |u http://dx.doi.org/10.1016/j.bbamcr.2023.119543 |3 Volltext |
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