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- PDB-7efl: Crystal structure of the gastric proton pump K791S in (BYK)E2BeF state -

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Basic information

Entry
Database: PDB / ID: 7efl
TitleCrystal structure of the gastric proton pump K791S 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 / intracellular potassium 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 sodium ion homeostasis / intracellular potassium 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: 3.4 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 1.3Nov 6, 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)139,31510
Polymers138,1972
Non-polymers1,1188
Water543
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5250 Å2
ΔGint-49 kcal/mol
Surface area52740 Å2
MethodPISA
Unit cell
Length a, b, c (Å)104.970, 104.970, 369.940
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: 109220.570 Da / Num. of mol.: 1 / Mutation: K791S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Sus scrofa (pig) / Gene: ATP4A / Cell line (production host): HEK293S GnT1- / 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: 28976.170 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Sus scrofa (pig) / Gene: ATP4B / Cell line (production host): HEK293S GnT1- / Production host: Homo sapiens (human) / References: UniProt: P18434

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Sugars , 1 types, 2 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.: 2 / 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, 9 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.: 3 / 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: 4.26 Å3/Da / Density % sol: 71.11 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 6.5
Details: 10% glycerol, 20% PEG 2000 MME, 0.2M KCl, 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 28, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 3.4→48.29 Å / Num. obs: 33564 / % possible obs: 99.8 % / Redundancy: 20 % / Biso Wilson estimate: 94.55 Å2 / CC1/2: 0.99 / Rmerge(I) obs: 0.165 / Rpim(I) all: 0.039 / Net I/σ(I): 12.4
Reflection shellResolution: 3.4→3.5 Å / Redundancy: 21.1 % / Rmerge(I) obs: 2.48 / Num. unique obs: 3274 / CC1/2: 0.79 / Rpim(I) all: 0.548 / % possible all: 99.8

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Processing

Software
NameVersionClassification
PHASER1.19_4092phasing
PHENIX1.19_4092refinement
XDSdata scaling
XDSdata reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5ylv

5ylv


Resolution: 3.4→48.29 Å / SU ML: 0.4588 / Cross valid method: FREE R-VALUE / Phase error: 32.3601
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2889 1661 4.95 %
Rwork0.23 31885 -
obs0.2329 33546 99.89 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 103.43 Å2
Refinement stepCycle: LAST / Resolution: 3.4→48.29 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9697 0 56 3 9756
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01179976
X-RAY DIFFRACTIONf_angle_d1.337613567
X-RAY DIFFRACTIONf_chiral_restr0.07431540
X-RAY DIFFRACTIONf_plane_restr0.00981746
X-RAY DIFFRACTIONf_dihedral_angle_d13.43093640
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.4-3.50.34441190.32422641X-RAY DIFFRACTION99.82
3.5-3.610.36581220.29072591X-RAY DIFFRACTION99.96
3.61-3.740.37451460.28322609X-RAY DIFFRACTION99.89
3.74-3.890.29191340.26792619X-RAY DIFFRACTION99.89
3.89-4.070.3181470.23872594X-RAY DIFFRACTION99.85
4.07-4.280.29911450.2232603X-RAY DIFFRACTION99.82
4.28-4.550.26421290.2032674X-RAY DIFFRACTION99.93
4.55-4.90.2811240.18722640X-RAY DIFFRACTION99.93
4.9-5.390.26781530.22882644X-RAY DIFFRACTION99.86
5.4-6.170.31031260.24132722X-RAY DIFFRACTION99.93
6.18-7.770.27881620.24722677X-RAY DIFFRACTION100
7.78-48.290.25821540.19682871X-RAY DIFFRACTION99.8
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)
10.559734311278-0.0063927211146-2.235463368580.4084052417980.7849929525060.7230839825290.13662103155-0.142036950476-0.10981836725-0.6012583587330.0453829093736-0.205112254677-0.303858011048-0.212067414449-0.06819769555581.213481270980.2817230131280.09676491433270.7436328662780.0444284706210.661585283389-55.387526323241.952176159329.3063456494
20.0842117570208-0.143571479296-0.979174114760.8156407522721.053865477362.3979042679-0.308231610075-0.218281864985-0.188209177393-0.109266484348-0.1184840134050.1515784802090.162381039374-0.01467284266870.3781352725730.8453013217560.2533094766350.1970277992350.6090091567410.07884469203490.744429625497-61.66128851337.637956417544.5176993747
3-0.1682201157820.0710927806712-0.163103943720.825678346203-0.2805325438032.405942366810.06978834238330.04754852227930.0163440152830.09917900650980.2066890175320.314654991153-0.346688350986-0.45894362693-0.1993864957710.6060947503050.2342701990720.2118457663640.599905938690.1541496109420.900005040925-63.874339702617.889595689442.066097941
40.183606387339-0.185140274691-0.3716181460431.4688559691-0.3836173882942.193200407830.0111183432288-0.2062630244620.1074133182640.2880982708810.07125293033-0.0213601691504-0.09494337947260.07870344336630.004029939204940.957631526430.2725242301020.1656952920130.6220332829940.1595230321270.737421566534-58.699867826918.211395664764.8907877379
50.2964088741960.5172889859290.2721064722660.36989074179-0.4820269055042.00423996646-0.2955248306440.160102579984-0.119090246059-0.09321475823970.165393531548-0.1130782953080.827170456169-0.1145248187450.1475312283161.11894165532-0.004282613043420.2689840554110.5277408335340.002955995166230.745359850589-54.87527249727.088346576124.49094765899
61.84378650132-0.0483148663697-1.086506023471.729053965870.7323090407612.062651834380.187920086178-0.406677897129-0.289874115845-0.343866598274-0.268358718194-0.3097341062861.42166227786-0.3280955154190.002369944317471.818467484880.048333024880.3644332905910.6721912416210.0876716233580.897890074191-59.3132125285-3.106756451226.20905572035
73.799955816680.5918931777781.403515588462.51573254669-0.801586390532.19940825699-0.442252719404-1.05256437735-0.661781356964-1.645328454770.153884695779-0.2921500165710.5389902937320.591601342418-0.1957052926831.562715501310.3234283244190.6594197742630.4245192409740.2749523598041.28214098214-41.7891180522-8.88601274081-2.54913415851
81.02974073554-0.0393845953854-0.5321534967091.61607405877-1.011927235960.666796844017-0.3349253164180.107735032433-0.3630898863710.003093755392470.00348624961148-0.0274180243261.18220334949-0.3302919902030.2391944881251.64551415232-0.3231005333780.2499062746811.0589205755-0.2668566068260.788077738488-55.18794564817.4428926116-43.4271084931
91.448258429570.4302254355070.5609661015351.29542287828-1.263906398842.47810610379-0.01039848781540.1163893747760.4961307904040.263704871599-0.1455415413180.6318159454740.44965615828-0.5155691998750.09615123811011.06862140756-0.3217716439450.2397700267561.60209687953-0.1141004851111.02647807311-69.302148661715.0683620174-45.1619945547
100.653386076917-0.483331528407-0.2380982227391.850863992160.01463167943690.557269863993-0.5175268383160.292415615888-0.1542903156370.4092843771710.1780050254810.606842987850.649414036431-0.5015279540080.2320628568341.49787241333-0.3082552689670.2877989774871.18026021065-0.2707985437060.637638227314-62.690577111813.2612802764-40.8573725272
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11chain 'A' and (resid 47 through 135 )AA47 - 1351 - 89
22chain 'A' and (resid 136 through 291 )AA136 - 29190 - 245
33chain 'A' and (resid 292 through 497 )AA292 - 497246 - 451
44chain 'A' and (resid 498 through 736 )AA498 - 736452 - 690
55chain 'A' and (resid 737 through 946 )AA737 - 946691 - 900
66chain 'A' and (resid 947 through 1033 )AA947 - 1033901 - 987
77chain 'B' and (resid 37 through 64 )BB37 - 641 - 28
88chain 'B' and (resid 65 through 185 )BB65 - 18529 - 149
99chain 'B' and (resid 186 through 222 )BB186 - 222150 - 186
1010chain 'B' and (resid 223 through 290 )BB223 - 290187 - 254

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