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- PDB-7x22: Cryo-EM structure of non gastric H,K-ATPase alpha2 K794S in (2K+)... -

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Basic information

Entry
Database: PDB / ID: 7x22
TitleCryo-EM structure of non gastric H,K-ATPase alpha2 K794S in (2K+)E2-AlF state
Components
  • Potassium-transporting ATPase alpha chain 2
  • Sodium/potassium-transporting ATPase subunit beta-1
KeywordsMEMBRANE PROTEIN / P-type ATPase / transporter / proton pump / kidney / colon / airway
Function / homology
Function and homology information


Basigin interactions / H+/K+-exchanging ATPase / Ion transport by P-type ATPases / P-type potassium:proton transporter activity / Na+/K+-exchanging ATPase / 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 ...Basigin interactions / H+/K+-exchanging ATPase / Ion transport by P-type ATPases / P-type potassium:proton transporter activity / Na+/K+-exchanging ATPase / 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 / potassium ion homeostasis / positive regulation of potassium ion transmembrane transport / regulation of calcium ion transmembrane transport / intracellular sodium ion homeostasis / response to metal ion / relaxation of cardiac muscle / regulation of cardiac muscle contraction by calcium ion signaling / Ion homeostasis / positive regulation of sodium ion transmembrane transport / sodium ion transport / organelle membrane / potassium ion import across plasma membrane / intracellular potassium ion homeostasis / ATPase activator activity / intercalated disc / blastocyst development / lateral plasma membrane / sperm flagellum / transporter activator activity / ATP metabolic process / cardiac muscle contraction / T-tubule / sodium ion transmembrane transport / proton transmembrane transport / protein localization to plasma membrane / sarcolemma / caveola / potassium ion transport / transmembrane transport / intracellular calcium ion homeostasis / ATPase binding / regulation of gene expression / protein-macromolecule adaptor activity / basolateral plasma membrane / response to hypoxia / cell adhesion / protein stabilization / apical plasma membrane / protein heterodimerization activity / innate immune response / protein kinase binding / ATP hydrolysis activity / ATP binding / metal ion binding / membrane / plasma membrane
Similarity search - Function
Sodium and potassium ATPases beta subunits signature 2. / Sodium/potassium-transporting ATPase subunit beta / Sodium/potassium-transporting ATPase subunit beta superfamily / Sodium / potassium ATPase beta chain / Sodium and potassium ATPases beta subunits signature 1. / : / P-type ATPase subfamily IIC, subunit alpha / Calcium-transporting ATPase, cytoplasmic domain N / Calcium-transporting ATPase, cytoplasmic domain N / Cation-transporting P-type ATPase, C-terminal ...Sodium and potassium ATPases beta subunits signature 2. / Sodium/potassium-transporting ATPase subunit beta / Sodium/potassium-transporting ATPase subunit beta superfamily / Sodium / potassium ATPase beta chain / Sodium and potassium ATPases beta subunits signature 1. / : / P-type ATPase subfamily IIC, subunit alpha / Calcium-transporting ATPase, cytoplasmic domain N / Calcium-transporting ATPase, cytoplasmic domain N / Cation-transporting P-type ATPase, C-terminal / Cation transporting ATPase, C-terminus / Cation transporter/ATPase, N-terminus / Cation-transporting P-type ATPase, N-terminal / Cation transporter/ATPase, N-terminus / P-type ATPase, cytoplasmic domain N / P-type ATPase actuator domain / P-type ATPase, haloacid dehalogenase domain / P-type ATPase, phosphorylation site / P-type ATPase, cytoplasmic domain N / E1-E2 ATPases phosphorylation site. / P-type ATPase, A domain superfamily / P-type ATPase / P-type ATPase, transmembrane domain superfamily / haloacid dehalogenase-like hydrolase / HAD superfamily / HAD-like superfamily / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
TETRAFLUOROALUMINATE ION / CHOLESTEROL / : / 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / Sodium/potassium-transporting ATPase subunit beta-1 / Potassium-transporting ATPase alpha chain 2
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsNakanishi, H. / Abe, K.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)21H02426 Japan
CitationJournal: Nat Commun / Year: 2022
Title: Structure and function of H/K pump mutants reveal Na/K pump mechanisms.
Authors: Victoria C Young / Hanayo Nakanishi / Dylan J Meyer / Tomohiro Nishizawa / Atsunori Oshima / Pablo Artigas / Kazuhiro Abe /
Abstract: Ion-transport mechanisms evolve by changing ion-selectivity, such as switching from Na to H selectivity in secondary-active transporters or P-type-ATPases. Here we study primary-active transport via ...Ion-transport mechanisms evolve by changing ion-selectivity, such as switching from Na to H selectivity in secondary-active transporters or P-type-ATPases. Here we study primary-active transport via P-type ATPases using functional and structural analyses to demonstrate that four simultaneous residue substitutions transform the non-gastric H/K pump, a strict H-dependent electroneutral P-type ATPase, into a bona fide Na-dependent electrogenic Na/K pump. Conversion of a H-dependent primary-active transporter into a Na-dependent one provides a prototype for similar studies of ion-transport proteins. Moreover, we solve the structures of the wild-type non-gastric H/K pump, a suitable drug target to treat cystic fibrosis, and of its Na/K pump-mimicking mutant in two major conformations, providing insight on how Na binding drives a concerted mechanism leading to Na/K pump phosphorylation.
History
DepositionFeb 25, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 5, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 13, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Potassium-transporting ATPase alpha chain 2
B: Sodium/potassium-transporting ATPase subunit beta-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)152,52116
Polymers146,5592
Non-polymers5,96214
Water181
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein Potassium-transporting ATPase alpha chain 2 / HK alpha 2 / Non-gastric H(+)/K(+) ATPase subunit alpha / Non-gastric Na(+)/K(+) ATPase subunit ...HK alpha 2 / Non-gastric H(+)/K(+) ATPase subunit alpha / Non-gastric Na(+)/K(+) ATPase subunit alpha / Proton pump / Sodium pump


Mass: 109042.586 Da / Num. of mol.: 1 / Mutation: K794S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Atp12a, Atp1al1 / Production host: Homo sapiens (human)
References: UniProt: P54708, H+/K+-exchanging ATPase, Na+/K+-exchanging ATPase
#2: Protein Sodium/potassium-transporting ATPase subunit beta-1 / Sodium/potassium-dependent ATPase subunit beta-1


Mass: 37516.859 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Atp1b1 / Production host: Homo sapiens (human) / References: UniProt: P07340

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Sugars , 1 types, 1 molecules

#8: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 6 types, 14 molecules

#3: Chemical ChemComp-ALF / TETRAFLUOROALUMINATE ION


Mass: 102.975 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: AlF4
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: K / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H46O
#7: Chemical
ChemComp-PCW / 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / (Z,Z)-4-HYDROXY-N,N,N-TRIMETHYL-10-OXO-7-[(1-OXO-9-OCTADECENYL)OXY]-3,5,9-TRIOXA-4-PHOSPHAHEPTACOS-18-EN-1-AMINIUM-4-OXIDE


Mass: 787.121 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C44H85NO8P / Comment: DOPC, phospholipid*YM
#9: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: non gastric H,K-ATPase / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 150 kDa/nm / Experimental value: YES
Source (natural)Organism: Rattus norvegicus (Norway rat)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 6.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 48 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.19_4092refinement
PHENIX1.19_4092refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 873901 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 100.27 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002510451
ELECTRON MICROSCOPYf_angle_d0.528514121
ELECTRON MICROSCOPYf_chiral_restr0.03951604
ELECTRON MICROSCOPYf_plane_restr0.00291770
ELECTRON MICROSCOPYf_dihedral_angle_d8.4621615

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