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- PDB-7efn: Crystal structure of the gastric proton pump K791S/E820D/Y340N/E9... -

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Basic information

Entry
Database: PDB / ID: 7efn
TitleCrystal structure of the gastric proton pump K791S/E820D/Y340N/E936V 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 sodium ion homeostasis / potassium ion import across plasma membrane / intracellular potassium ion homeostasis / 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 sodium ion homeostasis / potassium ion import across plasma membrane / intracellular potassium ion homeostasis / 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 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 / 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 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 / 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
Revision 1.3Oct 23, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _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: Sodium/potassium-transporting ATPase subunit alpha
B: Potassium-transporting ATPase subunit beta
hetero molecules


Theoretical massNumber of molelcules
Total (without water)139,90911
Polymers138,5702
Non-polymers1,3399
Water75742
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5560 Å2
ΔGint-55 kcal/mol
Surface area53680 Å2
MethodPISA
Unit cell
Length a, b, c (Å)105.940, 105.940, 372.010
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: 108939.234 Da / Num. of mol.: 1 / Mutation: K791S, E820D, Y340N, E936V
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 , 4 types, 48 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.: 4 / 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
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 42 / 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: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 4.35 Å3/Da / Density % sol: 71.72 %
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: Oct 15, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 3.2→48.71 Å / Num. obs: 41073 / % possible obs: 81.73 % / Redundancy: 2 % / Biso Wilson estimate: 58.03 Å2 / CC1/2: 1 / Rmerge(I) obs: 0.033 / Rpim(I) all: 0.033 / Net I/σ(I): 9.15
Reflection shellResolution: 3.2→3.3 Å / Rmerge(I) obs: 2.6 / Mean I/σ(I) obs: 0.28 / Num. unique obs: 1017 / CC1/2: 0.16 / Rpim(I) all: 2.6 / % possible all: 25.24

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Processing

Software
NameVersionClassification
PHENIX1.19_4092refinement
XDSdata reduction
XDSdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5ylv

5ylv


Resolution: 3.2→48.71 Å / SU ML: 0.3783 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 34.7919
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.3081 1620 4.82 %
Rwork0.2684 31974 -
obs0.2704 33594 81.77 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 60.14 Å2
Refinement stepCycle: LAST / Resolution: 3.2→48.71 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9733 0 70 42 9845
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.004210025
X-RAY DIFFRACTIONf_angle_d0.895113634
X-RAY DIFFRACTIONf_chiral_restr0.06281554
X-RAY DIFFRACTIONf_plane_restr0.00871753
X-RAY DIFFRACTIONf_dihedral_angle_d7.31621379
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.2-3.290.425340.3922782X-RAY DIFFRACTION24.16
3.29-3.40.3722350.32871177X-RAY DIFFRACTION36.11
3.4-3.520.3827920.32281647X-RAY DIFFRACTION52
3.52-3.660.44391150.34652457X-RAY DIFFRACTION76.37
3.66-3.830.37831520.32522897X-RAY DIFFRACTION90.58
3.83-4.030.37381670.3083179X-RAY DIFFRACTION99.02
4.03-4.280.27171620.25243231X-RAY DIFFRACTION100
4.28-4.610.25681560.23093279X-RAY DIFFRACTION99.85
4.61-5.080.28361470.23643263X-RAY DIFFRACTION99.97
5.08-5.810.28121710.26883274X-RAY DIFFRACTION99.94
5.81-7.320.29731910.27183298X-RAY DIFFRACTION99.97
7.32-48.710.2741980.23173490X-RAY DIFFRACTION99.84
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.216886808510.0001808929248420.148349853250.403471434175-0.4158638342211.061992509660.00544920579137-0.178915382028-0.0186295183519-0.286652463736-0.00273678217449-0.07386340921190.1707399690150.0198068968427-0.02773578663530.261745543337-0.0512144622698-0.1677212192430.1899359363-0.07946498239990.2702900719959.9162178983-39.043959038839.2124347144
20.3903804311390.09354316170120.3558554860220.3998438819740.2590953938591.05678117978-0.0322197121205-0.362691468476-0.02919031033770.2546717348660.209639748176-0.3263965381520.2322219657670.224451909438-0.1533664209170.215595033768-0.135320404208-0.3660310137350.129374822153-0.187378513910.33463282400267.934189699-18.506659604955.917651466
30.1296587164580.154918266419-0.04721646095450.118471400910.0999830950750.4442573339610.0363299663616-0.003213217490780.429329946962-0.06557213345940.007321134873810.275278297933-0.3989972595680.04086505638960.1763662629350.684099137923-0.329328341709-0.17629524736-0.0254647599687-0.1526103998950.34571718067953.3682272037-7.7029055687318.3181869171
44.26558796820.941584009553-2.213176744721.88218221861-0.4179445804282.517677541750.160993431434-0.02981034788140.3677180263240.295193732121-0.2903488447310.34867862369-0.0755212555888-0.4143221452880.1440822125060.804949116251-0.151099265681-0.3396947154270.482803408226-0.1680844011590.54162424499540.20193139027.35199824434-0.273612051742
50.7189201131720.364774898148-0.01096093494051.565829140680.1784877080320.257550011597-0.2427848770450.1523360883360.385379702928-0.1242616236210.03955384770770.0138502753201-0.4307411229550.2423753796010.01967401405310.582741687508-0.226914905555-0.0437060300010.2264170825190.1175822669320.31252147422156.2709163137-8.53845171331-41.145658794
60.6456536989210.256216165056-0.2723273068441.496625311390.1789827376350.7138927952230.04104982959240.1398623917420.327180278474-0.2261876695860.04689184656840.0746804608779-0.328576497252-0.00767921829467-0.0242207875230.533963740293-0.211536966617-0.1268063636550.2863714404180.2130376471920.32657680100958.5957142427-5.21056658678-47.0440417848
74.850706724220.622126492074-0.2604163526330.4612471182541.537451597657.04633863870.0455843241792-0.173694497874-0.8773297926420.2990526227580.0820686580256-0.5826675529070.910520943130.645155954549-0.1301525004110.790144900830.0245844054043-0.196919827870.6067098074170.0481904559670.56992043374171.683353553-25.7461173759-45.5629796404
80.168953730429-0.002499784469330.05940408585791.19827799691-0.1719097183770.402651942283-0.03079388418710.01867922584880.06749262496860.094360224564-0.023592450494-0.129292595242-0.3037724142450.2685372606090.07348996995290.67096204026-0.178351383659-0.1155674785540.4782566389120.2018415437470.16500944019863.851549765-13.3421815238-42.3410741788
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11chain 'A' and (resid 49 through 291 )AA49 - 2911 - 243
22chain 'A' and (resid 292 through 602 )AA292 - 602244 - 554
33chain 'A' and (resid 603 through 1033 )AA603 - 1033555 - 985
44chain 'B' and (resid 30 through 65 )BE30 - 651 - 36
55chain 'B' and (resid 66 through 140 )BE66 - 14037 - 111
66chain 'B' and (resid 141 through 197 )BE141 - 197112 - 168
77chain 'B' and (resid 198 through 214 )BE198 - 214169 - 185
88chain 'B' and (resid 215 through 290 )BE215 - 290186 - 261

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