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- PDB-8zyj: Cryo-EM structure of human testis-specific Na+,K+-ATPase alpha4 i... -

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Basic information

Entry
Database: PDB / ID: 8zyj
TitleCryo-EM structure of human testis-specific Na+,K+-ATPase alpha4 in ouabain-bound form
Components(Sodium/potassium-transporting ATPase subunit ...) x 2
KeywordsTRANSPORT PROTEIN / P-type ATPase / sodium pump / Na+ / K+-ATPase / testis / human
Function / homology
Function and homology information


photoreceptor cell cilium / protein transport into plasma membrane raft / Na+/K+-exchanging ATPase / positive regulation of sodium ion export across plasma membrane / positive regulation of potassium ion import across plasma membrane / membrane repolarization during cardiac muscle cell action potential / rod photoreceptor outer segment / P-type sodium:potassium-exchanging transporter activity / sodium:potassium-exchanging ATPase complex / membrane repolarization ...photoreceptor cell cilium / protein transport into plasma membrane raft / Na+/K+-exchanging ATPase / positive regulation of sodium ion export across plasma membrane / positive regulation of potassium ion import across plasma membrane / membrane repolarization during cardiac muscle cell action potential / rod photoreceptor outer segment / P-type sodium:potassium-exchanging transporter activity / sodium:potassium-exchanging ATPase complex / membrane repolarization / establishment or maintenance of transmembrane electrochemical gradient / cell communication by electrical coupling involved in cardiac conduction / sodium ion export across plasma membrane / flagellated sperm motility / ATPase-coupled monoatomic cation transmembrane transporter activity / intracellular sodium ion homeostasis / regulation of calcium ion transmembrane transport / fertilization / regulation of cellular pH / relaxation of cardiac muscle / regulation of cardiac muscle contraction by calcium ion signaling / Basigin interactions / sodium ion transport / organelle membrane / potassium ion import across plasma membrane / intracellular potassium ion homeostasis / ATPase activator activity / Ion transport by P-type ATPases / intercalated disc / lateral plasma membrane / sperm flagellum / transporter activator activity / transport across blood-brain barrier / ATP metabolic process / cardiac muscle contraction / Ion homeostasis / monoatomic ion transport / sperm midpiece / potassium ion transmembrane transport / T-tubule / sodium ion transmembrane transport / proton transmembrane transport / protein localization to plasma membrane / regulation of membrane potential / cell projection / establishment of localization in cell / sarcolemma / caveola / potassium ion transport / kinase binding / intracellular calcium ion homeostasis / MHC class II protein complex binding / extracellular vesicle / ATPase binding / regulation of gene expression / spermatogenesis / protein-macromolecule adaptor activity / basolateral plasma membrane / Potential therapeutics for SARS / response to hypoxia / cell surface receptor signaling pathway / cell adhesion / protein stabilization / apical plasma membrane / membrane raft / protein heterodimerization activity / innate immune response / protein kinase binding / ATP hydrolysis activity / extracellular exosome / ATP binding / metal ion binding / membrane / plasma membrane
Similarity search - Function
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 ...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, 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
Similarity search - Domain/homology
CHOLESTEROL / OUABAIN / 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / Chem-Q7G / Sodium/potassium-transporting ATPase subunit beta-1 / Sodium/potassium-transporting ATPase subunit alpha-4
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.37 Å
AuthorsAbe, K. / Blanco, G.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)24K01975 Japan
CitationJournal: Int J Mol Sci / Year: 2024
Title: Molecular Structure of the Na,K-ATPase α4β1 Isoform in Its Ouabain-Bound Conformation.
Authors: Kazuhiro Abe / Jeff McDermott / Hridya Valia Madapally / Parthiban Marimuthu / Chai C Gopalasingam / Christoph Gerle / Hideki Shigematsu / Himanshu Khandelia / Gustavo Blanco /
Abstract: Na,K-ATPase is the active ion transport system that maintains the electrochemical gradients for Na and K across the plasma membrane of most animal cells. Na,K-ATPase is constituted by the association ...Na,K-ATPase is the active ion transport system that maintains the electrochemical gradients for Na and K across the plasma membrane of most animal cells. Na,K-ATPase is constituted by the association of two major subunits, a catalytic α and a glycosylated β subunit, both of which exist as different isoforms (in mammals known as α1, α2, α3, α4, β1, β2 and β3). Na,K-ATPase α and β isoforms assemble in different combinations to produce various isozymes with tissue specific expression and distinct biochemical properties. Na,K-ATPase α4β1 is only found in male germ cells of the testis and is mainly expressed in the sperm flagellum, where it plays a critical role in sperm motility and male fertility. Here, we report the molecular structure of Na,K-ATPase α4β1 at 2.37 Å resolution in the ouabain-bound state and in the presence of beryllium fluoride. Overall, Na,K-ATPase α4 structure exhibits the basic major domains of a P-Type ATPase, resembling Na,K-ATPase α1, but has differences specific to its distinct sequence. Dissimilarities include the site where the inhibitor ouabain binds. Molecular simulations indicate that glycosphingolipids can bind to a putative glycosphingolipid binding site, which could potentially modulate Na,K-ATPase α4 activity. This is the first experimental evidence for the structure of Na,K-ATPase α4β1. These data provide a template that will aid in better understanding the function Na,K-ATPase α4β1 and will be important for the design and development of compounds that can modulate Na,K-ATPase α4 activity for the purpose of improving male fertility or to achieve male contraception.
History
DepositionJun 18, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 4, 2024Provider: repository / Type: Initial release
Revision 1.1Dec 11, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Sodium/potassium-transporting ATPase subunit alpha-4
B: Sodium/potassium-transporting ATPase subunit beta-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)149,85112
Polymers144,1072
Non-polymers5,74410
Water5,441302
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Sodium/potassium-transporting ATPase subunit ... , 2 types, 2 molecules AB

#1: Protein Sodium/potassium-transporting ATPase subunit alpha-4 / Na(+)/K(+) ATPase alpha-4 subunit / Sodium pump subunit alpha-4


Mass: 108998.727 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ATP1A4, ATP1AL2 / Production host: Homo sapiens (human) / References: UniProt: Q13733, Na+/K+-exchanging ATPase
#2: Protein Sodium/potassium-transporting ATPase subunit beta-1 / Sodium/potassium-dependent ATPase subunit beta-1


Mass: 35108.258 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ATP1B1, ATP1B / Production host: Homo sapiens (human) / References: UniProt: P05026

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Sugars , 1 types, 2 molecules

#9: 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 , 7 types, 310 molecules

#3: Chemical ChemComp-MG / MAGNESIUM ION


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


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
#5: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H46O
#6: Chemical ChemComp-OBN / OUABAIN


Mass: 584.652 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C29H44O12 / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-Q7G / 2-{[(4-O-alpha-D-glucopyranosyl-alpha-D-glucopyranosyl)oxy]methyl}-4-{[(3beta,9beta,14beta,17beta,25R)-spirost-5-en-3-yl]oxy}butyl 4-O-alpha-D-glucopyranosyl-alpha-D-glucopyranoside


Mass: 1165.315 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C56H92O25 / Feature type: SUBJECT OF INVESTIGATION
#8: 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.: 1 / Source method: obtained synthetically / Formula: C44H85NO8P / Comment: DOPC, phospholipid*YM
#10: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 302 / 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: alpha4-beta1 heterodimer / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 135 kDa/nm / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 6.5
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 10000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.37 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 162837 / Symmetry type: POINT
RefinementCross valid method: NONE

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