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- PDB-7efm: Crystal structure of the gastric proton pump K791S/E820D/Y340N in... -

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Basic information

Entry
Database: PDB / ID: 7efm
TitleCrystal structure of the gastric proton pump K791S/E820D/Y340N in (BYK)E2BeF state
Components
  • Potassium-transporting ATPase subunit beta
  • Sodium/potassium-transporting ATPase subunit alpha
KeywordsMEMBRANE PROTEIN / Cation pump / P-type ATPase / gastric / proton pump
Function / homology
Function and homology information


potassium:proton exchanging ATPase complex / P-type potassium:proton transporter activity / Ion transport by P-type ATPases / sodium:potassium-exchanging ATPase complex / sodium ion export across plasma membrane / intracellular potassium ion homeostasis / intracellular sodium ion homeostasis / potassium ion import across plasma membrane / ATPase activator activity / potassium ion transmembrane transport ...potassium:proton exchanging ATPase complex / P-type potassium:proton transporter activity / Ion transport by P-type ATPases / sodium:potassium-exchanging ATPase complex / sodium ion export across plasma membrane / intracellular potassium ion homeostasis / intracellular sodium ion homeostasis / potassium ion import across plasma membrane / ATPase activator activity / potassium ion transmembrane transport / proton transmembrane transport / cell adhesion / apical plasma membrane / magnesium ion binding / ATP hydrolysis activity / ATP binding
Similarity search - Function
Gastric H+/K+-transporter P-type ATPase, N-terminal / Gastric H+/K+-ATPase, N terminal domain / 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. / Sodium and potassium ATPases beta subunits signature 2. / P-type ATPase subfamily IIC, subunit alpha / Cation-transporting P-type ATPase, C-terminal / Cation transporting ATPase, C-terminus ...Gastric H+/K+-transporter P-type ATPase, N-terminal / Gastric H+/K+-ATPase, N terminal domain / 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. / Sodium and potassium ATPases beta subunits signature 2. / P-type ATPase subfamily IIC, subunit alpha / 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, 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 / HAD superfamily / HAD-like superfamily
Similarity search - Domain/homology
Chem-J3C / RUBIDIUM ION / Sodium/potassium-transporting ATPase subunit alpha / Potassium-transporting ATPase subunit beta
Similarity search - Component
Biological speciesSus scrofa (pig)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.2 Å
AuthorsAbe, K. / Yamamoto, K. / Irie, K.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)17H03653 Japan
CitationJournal: Nat Commun / Year: 2021
Title: Gastric proton pump with two occluded K engineered with sodium pump-mimetic mutations.
Authors: Kazuhiro Abe / Kenta Yamamoto / Katsumasa Irie / Tomohiro Nishizawa / Atsunori Oshima /
Abstract: The gastric H,K-ATPase mediates electroneutral exchange of 1H/1K per ATP hydrolysed across the membrane. Previous structural analysis of the K-occluded E2-P transition state of H,K-ATPase showed a ...The gastric H,K-ATPase mediates electroneutral exchange of 1H/1K per ATP hydrolysed across the membrane. Previous structural analysis of the K-occluded E2-P transition state of H,K-ATPase showed a single bound K at cation-binding site II, in marked contrast to the two K ions occluded at sites I and II of the closely-related Na,K-ATPase which mediates electrogenic 3Na/2K translocation across the membrane. The molecular basis of the different K stoichiometry between these K-counter-transporting pumps is elusive. We show a series of crystal structures and a cryo-EM structure of H,K-ATPase mutants with changes in the vicinity of site I, based on the structure of the sodium pump. Our step-wise and tailored construction of the mutants finally gave a two-K bound H,K-ATPase, achieved by five mutations, including amino acids directly coordinating K (Lys791Ser, Glu820Asp), indirectly contributing to cation-binding site formation (Tyr340Asn, Glu936Val), and allosterically stabilizing K-occluded conformation (Tyr799Trp). This quintuple mutant in the K-occluded E2-P state unambiguously shows two separate densities at the cation-binding site in its 2.6 Å resolution cryo-EM structure. These results offer new insights into how two closely-related cation pumps specify the number of K accommodated at their cation-binding site.
History
DepositionMar 22, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 1, 2021Provider: repository / Type: Initial release
Revision 1.1Mar 16, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Nov 29, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)140,04210
Polymers138,7882
Non-polymers1,2548
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5250 Å2
ΔGint-50 kcal/mol
Surface area53660 Å2
MethodPISA
Unit cell
Length a, b, c (Å)104.690, 104.690, 368.020
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number152
Space group name H-MP3121
Space group name HallP312"
Symmetry operation#1: x,y,z
#2: -y,x-y,z+1/3
#3: -x+y,-x,z+2/3
#4: x-y,-y,-z+2/3
#5: -x,-x+y,-z+1/3
#6: y,x,-z

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein Sodium/potassium-transporting ATPase subunit alpha


Mass: 109157.469 Da / Num. of mol.: 1 / Mutation: K791S, E820D, Y340N
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Sus scrofa (pig) / Gene: ATP4A / Production host: Homo sapiens (human) / References: UniProt: A0A5G2QYH2
#2: Protein Potassium-transporting ATPase subunit beta / Gastric H(+)/K(+) ATPase subunit beta / Proton pump beta chain / gp60-90


Mass: 29630.912 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Sus scrofa (pig) / Gene: ATP4B / Production host: Homo sapiens (human) / References: UniProt: P18434

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Sugars , 1 types, 3 molecules

#6: 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.: 3 / 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 , 3 types, 5 molecules

#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#4: Chemical ChemComp-RB / RUBIDIUM ION


Mass: 85.468 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Rb
#5: Chemical ChemComp-J3C / (7R,8R,9S)-2,3-dimethyl-9-phenyl-7,8,9,10-tetrahydroimidazo[1,2-h][1,7]naphthyridine-7,8-diol


Mass: 309.362 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C18H19N3O2 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 4.19 Å3/Da / Density % sol: 70.68 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 6.5
Details: 10% glycerol, 20% PEG 2000 MME, 0.2M RbCl, 3% methylpentanediol, 5mM beta-mercaptoethanol

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Data collection

DiffractionMean temperature: 80 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SPring-8 / Beamline: BL41XU / Wavelength: 1 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Nov 22, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 3.2→48.15 Å / Num. obs: 39699 / % possible obs: 94.8 % / Redundancy: 20.1 % / Biso Wilson estimate: 82.16 Å2 / CC1/2: 0.99 / Rmerge(I) obs: 0.11 / Rpim(I) all: 0.026 / Net I/σ(I): 16.8
Reflection shellResolution: 3.2→3.3 Å / Redundancy: 21.4 % / Rmerge(I) obs: 2.58 / Mean I/σ(I) obs: 1.6 / Num. unique obs: 2203 / CC1/2: 0.85 / Rpim(I) all: 0.57 / % possible all: 56.6

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Processing

Software
NameVersionClassification
PHENIX1.17.1_3660refinement
XDSdata reduction
XDSdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5ylv

5ylv


Resolution: 3.2→48.15 Å / SU ML: 0.4621 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 31.677
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2927 1769 4.7 %
Rwork0.2278 35908 -
obs0.231 37677 94.84 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 83.45 Å2
Refinement stepCycle: LAST / Resolution: 3.2→48.15 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9747 0 69 0 9816
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.010310039
X-RAY DIFFRACTIONf_angle_d1.334313651
X-RAY DIFFRACTIONf_chiral_restr0.08021554
X-RAY DIFFRACTIONf_plane_restr0.00821756
X-RAY DIFFRACTIONf_dihedral_angle_d19.99183667
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.2-3.290.4183540.32631537X-RAY DIFFRACTION52.87
3.29-3.380.3506940.29412318X-RAY DIFFRACTION80.29
3.38-3.490.35671320.28572884X-RAY DIFFRACTION99.9
3.49-3.620.34081350.26642859X-RAY DIFFRACTION99.93
3.62-3.760.28321420.25272852X-RAY DIFFRACTION99.93
3.76-3.930.28891510.23672861X-RAY DIFFRACTION99.9
3.93-4.140.28851400.21772876X-RAY DIFFRACTION99.9
4.14-4.40.27321510.20792889X-RAY DIFFRACTION99.87
4.4-4.740.2511230.19342920X-RAY DIFFRACTION99.8
4.74-5.210.25191420.19362921X-RAY DIFFRACTION99.8
5.22-5.970.3471470.23182917X-RAY DIFFRACTION99.93
5.97-7.510.3181790.24882969X-RAY DIFFRACTION100
7.52-48.150.27081790.20723105X-RAY DIFFRACTION99.7

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