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Yorodumi- PDB-8ijm: Cyo-EM structure of K794A non-gastric proton pump in Na+ bound E1... -
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-Basic information
Entry | Database: PDB / ID: 8ijm | ||||||
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Title | Cyo-EM structure of K794A non-gastric proton pump in Na+ bound E1AMPPCP state | ||||||
Components | (Sodium/potassium-transporting ATPase subunit ...) x 2 | ||||||
Keywords | MEMBRANE PROTEIN / P-type ATPase / P2-type ATPase | ||||||
Function / homology | Function and homology information Basigin interactions / Ion transport by P-type ATPases / P-type potassium:proton transporter activity / positive regulation of sodium ion export across plasma membrane / positive regulation of potassium ion import across plasma membrane / metal ion transport / P-type sodium:potassium-exchanging transporter activity / sodium:potassium-exchanging ATPase complex / membrane repolarization / regulation of pH ...Basigin interactions / Ion transport by P-type ATPases / P-type potassium:proton transporter activity / positive regulation of sodium ion export across plasma membrane / positive regulation of potassium ion import across plasma membrane / metal ion transport / P-type sodium:potassium-exchanging transporter activity / sodium:potassium-exchanging ATPase complex / membrane repolarization / regulation of pH / sodium ion export across plasma membrane / regulation of calcium ion transmembrane transport / intracellular potassium ion homeostasis / potassium ion homeostasis / intracellular sodium ion homeostasis / regulation of cardiac muscle contraction by calcium ion signaling / relaxation of cardiac muscle / Ion homeostasis / sodium ion transport / organelle membrane / potassium ion import across plasma membrane / monoatomic cation transmembrane transport / ATPase activator activity / blastocyst development / intercalated disc / lateral plasma membrane / sperm flagellum / sodium ion transmembrane transport / cardiac muscle contraction / ATP metabolic process / T-tubule / protein localization to plasma membrane / potassium ion transport / caveola / sarcolemma / intracellular calcium ion homeostasis / protein-macromolecule adaptor activity / ATPase binding / regulation of gene expression / basolateral plasma membrane / response to hypoxia / cell adhesion / protein stabilization / protein heterodimerization activity / apical plasma membrane / innate immune response / protein kinase binding / ATP hydrolysis activity / ATP binding / membrane / metal ion binding / plasma membrane Similarity search - Function | ||||||
Biological species | Rattus (rat) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.13 Å | ||||||
Authors | Abe, K. | ||||||
Funding support | Japan, 1items
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Citation | Journal: Biochim Biophys Acta Mol Cell Res / Year: 2023 Title: An unusual conformation from Na-sensitive non-gastric proton pump mutants reveals molecular mechanisms of cooperative Na-binding. Authors: Kazuhiro Abe / Tomohiro Nishizawa / Pablo Artigas / 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. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8ijm.cif.gz | 315.8 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8ijm.ent.gz | 203.7 KB | Display | PDB format |
PDBx/mmJSON format | 8ijm.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8ijm_validation.pdf.gz | 1.3 MB | Display | wwPDB validaton report |
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Full document | 8ijm_full_validation.pdf.gz | 1.4 MB | Display | |
Data in XML | 8ijm_validation.xml.gz | 51.6 KB | Display | |
Data in CIF | 8ijm_validation.cif.gz | 75.9 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ij/8ijm ftp://data.pdbj.org/pub/pdb/validation_reports/ij/8ijm | HTTPS FTP |
-Related structure data
Related structure data | 35489MC 8ijlC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Sodium/potassium-transporting ATPase subunit ... , 2 types, 2 molecules AB
#1: Protein | Mass: 109084.625 Da / Num. of mol.: 1 / Mutation: G315D, D512N, K794A Source method: isolated from a genetically manipulated source Source: (gene. exp.) Rattus (rat) / Gene: Atp12a / Cell line (production host): HEK293S GnT1- / Production host: Homo sapiens (human) / References: UniProt: G3V8S4 |
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#2: Protein | Mass: 37516.859 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Rattus (rat) / Gene: Atp1b1 / Cell line (production host): HEK293S GnT1- / Production host: Homo sapiens (human) / References: UniProt: P07340 |
-Sugars , 1 types, 1 molecules
#7: Sugar | ChemComp-NAG / |
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-Non-polymers , 4 types, 6 molecules
#3: Chemical | ChemComp-ACP / | ||||
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#4: Chemical | #5: Chemical | ChemComp-CLR / | #6: Chemical | |
-Details
Has ligand of interest | Y |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: hetero dimer of alpha and beta subunit / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT |
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Molecular weight | Value: 135 kDa/nm / Experimental value: YES |
Source (natural) | Organism: Rattus (rat) |
Source (recombinant) | Organism: Homo sapiens (human) |
Buffer solution | pH: 6.5 |
Specimen | Conc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm |
Image recording | Electron dose: 53 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
-Processing
Software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.13 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 198900 / Symmetry type: POINT | ||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||
Displacement parameters | Biso mean: 37.84 Å2 | ||||||||||||||||||||||||
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