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- EMDB-60570: Cryo-EM structure of human testis-specific Na+,K+-ATPase alpha4 i... -

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Entry
Database: EMDB / ID: EMD-60570
TitleCryo-EM structure of human testis-specific Na+,K+-ATPase alpha4 in ouabain-bound form
Map data
Sample
  • Complex: alpha4-beta1 heterodimer
    • Protein or peptide: Sodium/potassium-transporting ATPase subunit alpha-4
    • Protein or peptide: Sodium/potassium-transporting ATPase subunit beta-1
  • Ligand: MAGNESIUM ION
  • Ligand: SODIUM ION
  • Ligand: CHOLESTEROL
  • Ligand: OUABAIN
  • Ligand: 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
  • Ligand: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: water
KeywordsP-type ATPase / sodium pump / Na+ / K+-ATPase / testis / human / TRANSPORT PROTEIN
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 / transporter activator activity / rod photoreceptor outer segment / P-type sodium:potassium-exchanging transporter activity / sodium:potassium-exchanging ATPase complex ...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 / transporter activator activity / 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 / flagellated sperm motility / sodium ion export across plasma membrane / cell communication by electrical coupling involved in cardiac conduction / regulation of calcium ion transmembrane transport / ATPase-coupled monoatomic cation transmembrane transporter activity / intracellular sodium ion homeostasis / intracellular potassium ion homeostasis / fertilization / regulation of cellular pH / regulation of cardiac muscle contraction by calcium ion signaling / relaxation of cardiac muscle / Basigin interactions / sodium ion transport / organelle membrane / potassium ion import across plasma membrane / ATPase activator activity / Ion transport by P-type ATPases / sperm flagellum / intercalated disc / lateral plasma membrane / transport across blood-brain barrier / ATP metabolic process / Ion homeostasis / monoatomic ion transport / cardiac muscle contraction / potassium ion transmembrane transport / T-tubule / sperm midpiece / sodium ion transmembrane transport / proton transmembrane transport / protein localization to plasma membrane / regulation of membrane potential / cell projection / establishment of localization in cell / potassium ion transport / sarcolemma / kinase binding / intracellular calcium ion homeostasis / MHC class II protein complex binding / extracellular vesicle / ATPase binding / regulation of gene expression / protein-macromolecule adaptor activity / basolateral plasma membrane / spermatogenesis / Potential therapeutics for SARS / 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 / Cation transport ATPase (P-type) / E1-E2 ATPase / 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
Sodium/potassium-transporting ATPase subunit beta-1 / Sodium/potassium-transporting ATPase subunit alpha-4
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.37 Å
AuthorsAbe K / Blanco G
Funding support Japan, 1 items
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, 2024-
Header (metadata) releaseDec 4, 2024-
Map releaseDec 4, 2024-
UpdateDec 11, 2024-
Current statusDec 11, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_60570.png

Downloads & links

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Map

FileDownload / File: emd_60570.map.gz / Format: CCP4 / Size: 347.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.75 Å/pix.
x 450 pix.
= 338.4 Å		0.75 Å/pix.
x 450 pix.
= 338.4 Å		0.75 Å/pix.
x 450 pix.
= 338.4 Å

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.752 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.886
Minimum - Maximum-2.275366 - 4.372786
Average (Standard dev.)-0.0024274038 (±0.0760784)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions450450450
Spacing450450450
CellA=B=C: 338.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_60570_msk_1.map
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Half map: #2

Fileemd_60570_half_map_1.map
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Half map: #1

Fileemd_60570_half_map_2.map
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Sample components

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Entire : alpha4-beta1 heterodimer

EntireName: alpha4-beta1 heterodimer
Components
  • Complex: alpha4-beta1 heterodimer
    • Protein or peptide: Sodium/potassium-transporting ATPase subunit alpha-4
    • Protein or peptide: Sodium/potassium-transporting ATPase subunit beta-1
  • Ligand: MAGNESIUM ION
  • Ligand: SODIUM ION
  • Ligand: CHOLESTEROL
  • Ligand: OUABAIN
  • Ligand: 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
  • Ligand: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: water

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Supramolecule #1: alpha4-beta1 heterodimer

SupramoleculeName: alpha4-beta1 heterodimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 135 kDa/nm

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Macromolecule #1: Sodium/potassium-transporting ATPase subunit alpha-4

MacromoleculeName: Sodium/potassium-transporting ATPase subunit alpha-4 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: Na+/K+-exchanging ATPase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 108.998727 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MVVMDDHKLT LEELSTKYSV DLTKGHSHQR AKEILTRGGP NTVTPPPTTP EWVKFCKQLF GGFSLLLWTG AILCFVAYSI QIYFNEEPT KDNLYLSIVL SVVVIVTGCF SYYQEAKSSK IMESFKNMVP QQALVIRGGE KMQINVQEVV LGDLVEIKGG D RVPADLRL ...String:
MVVMDDHKLT LEELSTKYSV DLTKGHSHQR AKEILTRGGP NTVTPPPTTP EWVKFCKQLF GGFSLLLWTG AILCFVAYSI QIYFNEEPT KDNLYLSIVL SVVVIVTGCF SYYQEAKSSK IMESFKNMVP QQALVIRGGE KMQINVQEVV LGDLVEIKGG D RVPADLRL ISAQGCKVDN SSLTGESEPQ SRSPDFTHEN PLETRNICFF STNCVEGTAR GIVIATGDST VMGRIASLTS GL AVGQTPI AAEIEHFIHL ITVVAVFLGV TFFALSLLLG YGWLEAIIFL IGIIVANVPE GLLATVTVCL TLTAKRMARK NCL VKNLEA VETLGSTSTI CS(BFD)KTGTLTQ NRMTVAHMWF DMTVYEADTT EEQTGKTFTK SSDTWFMLAR IAGLCNRADF KANQEILPI AKRATTGDAS ESALLKFIEQ SYSSVAEMRE KNPKVAEIPF NSTNKYQMSI HLREDSSQTH VLMMKGAPER I LEFCSTFL LNGQEYSMND EMKEAFQNAY LELGGLGERV LGFCFLNLPS SFSKGFPFNT DEINFPMDNL CFVGLISMID PP RAAVPDA VSKCRSAGIK VIMVTGDHPI TAKAIAKGVG IISEGTETAE EVAARLKIPI SKVDASAAKA IVVHGAELKD IQS KQLDQI LQNHPEIVFA RTSPQQKLII VEGCQRLGAV VAVTGDGVND SPALKKADIG IAMGISGSDV SKQAADMILL DDNF ASIVT GVEEGRLIFD NLKKSIMYTL TSNIPEITPF LMFIILGIPL PLGTITILCI DLGTDMVPAI SLAYESAESD IMKRL PRNP KTDNLVNHRL IGMAYGQIGM IQALAGFFTY FVILAENGFR PVDLLGIRLH WEDKYLNDLE DSYGQQWTYE QRKVVE FTC QTAFFVTIVV VQWADLIISK TRRNSLFQQG MRNKVLIFGI LEETLLAAFL SYTPGMDVAL RMYPLKITWW LCAIPYS IL IFVYDEIRKL LIRQHPDGWV ERETYY

UniProtKB: Sodium/potassium-transporting ATPase subunit alpha-4

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Macromolecule #2: Sodium/potassium-transporting ATPase subunit beta-1

MacromoleculeName: Sodium/potassium-transporting ATPase subunit beta-1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 35.108258 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MARGKAKEEG SWKKFIWNSE KKEFLGRTGG SWFKILLFYV IFYGCLAGIF IGTIQVMLLT ISEFKPTYQD RVAPPGLTQI PQIQKTEIS FRPNDPKSYE AYVLNIVRFL EKYKDSAQRD DMIFEDCGDV PSEPKERGDF NHERGERKVC RFKLEWLGNC S GLNDETYG ...String:
MARGKAKEEG SWKKFIWNSE KKEFLGRTGG SWFKILLFYV IFYGCLAGIF IGTIQVMLLT ISEFKPTYQD RVAPPGLTQI PQIQKTEIS FRPNDPKSYE AYVLNIVRFL EKYKDSAQRD DMIFEDCGDV PSEPKERGDF NHERGERKVC RFKLEWLGNC S GLNDETYG YKEGKPCIII KLNRVLGFKP KPPKNESLET YPVMKYNPNV LPVQCTGKRD EDKDKVGNVE YFGLGNSPGF PL QYYPYYG KLLQPKYLQP LLAVQFTNLT MDTEIRIECK AYGENIGYSE KDRFQGRFDV KIEVKS

UniProtKB: Sodium/potassium-transporting ATPase subunit beta-1

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #4: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 4 / Number of copies: 1
Molecular weightTheoretical: 22.99 Da

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Macromolecule #5: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 5 / Number of copies: 1 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL

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Macromolecule #6: OUABAIN

MacromoleculeName: OUABAIN / type: ligand / ID: 6 / Number of copies: 1 / Formula: OBN
Molecular weightTheoretical: 584.652 Da
Chemical component information

ChemComp-OBN:
OUABAIN

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Macromolecule #7: 2-{[(4-O-alpha-D-glucopyranosyl-alpha-D-glucopyranosyl)oxy]methyl...

MacromoleculeName: 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
type: ligand / ID: 7 / Number of copies: 3 / Formula: Q7G
Molecular weightTheoretical: 1.165315 KDa
Chemical component information

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

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Macromolecule #8: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE

MacromoleculeName: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / type: ligand / ID: 8 / Number of copies: 1 / Formula: PCW
Molecular weightTheoretical: 787.121 Da
Chemical component information

ChemComp-PCW:
1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / DOPC, phospholipid*YM

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Macromolecule #9: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 9 / Number of copies: 2 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #10: water

MacromoleculeName: water / type: ligand / ID: 10 / Number of copies: 302 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration10 mg/mL
BufferpH: 6.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 10.0 µm / Nominal defocus min: 1.0 µm

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7wyt

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.37 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 162837
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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