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- PDB-8ox9: Cryo-EM structure of ATP8B1-CDC50A in E2P active conformation wit... -

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Basic information

Entry
Database: PDB / ID: 8ox9
TitleCryo-EM structure of ATP8B1-CDC50A in E2P active conformation with bound PC
Components
  • Cell cycle control protein 50A
  • Phospholipid-transporting ATPase IC
KeywordsMEMBRANE PROTEIN / lipid transporter autoinhibition P-type ATPase P4-ATPase CDC50A
Function / homology
Function and homology information


vestibulocochlear nerve formation / regulation of plasma membrane organization / regulation of microvillus assembly / positive regulation of phospholipid translocation / phosphatidylcholine flippase activity / aminophospholipid transport / aminophospholipid flippase activity / phosphatidylserine flippase activity / protein localization to endosome / inner ear receptor cell development ...vestibulocochlear nerve formation / regulation of plasma membrane organization / regulation of microvillus assembly / positive regulation of phospholipid translocation / phosphatidylcholine flippase activity / aminophospholipid transport / aminophospholipid flippase activity / phosphatidylserine flippase activity / protein localization to endosome / inner ear receptor cell development / ATPase-coupled intramembrane lipid transporter activity / phospholipid-translocating ATPase complex / phosphatidylserine floppase activity / positive regulation of protein exit from endoplasmic reticulum / xenobiotic transmembrane transport / phosphatidylcholine floppase activity / stereocilium / apical protein localization / bile acid metabolic process / P-type phospholipid transporter / cardiolipin binding / phospholipid translocation / bile acid and bile salt transport / azurophil granule membrane / transport vesicle membrane / Golgi organization / Ion transport by P-type ATPases / specific granule membrane / regulation of chloride transport / sensory perception of sound / trans-Golgi network / positive regulation of neuron projection development / late endosome membrane / early endosome membrane / monoatomic ion transmembrane transport / nuclear body / apical plasma membrane / negative regulation of DNA-templated transcription / Neutrophil degranulation / structural molecule activity / Golgi apparatus / magnesium ion binding / endoplasmic reticulum / ATP hydrolysis activity / nucleoplasm / ATP binding / membrane / plasma membrane / cytosol
Similarity search - Function
CDC50/LEM3 family / LEM3 (ligand-effect modulator 3) family / CDC50 family / P-type ATPase, subfamily IV / P-type ATPase, C-terminal / P-type ATPase, N-terminal / Phospholipid-translocating ATPase N-terminal / Phospholipid-translocating P-type ATPase C-terminal / Cation transport ATPase (P-type) / E1-E2 ATPase / P-type ATPase, haloacid dehalogenase domain ...CDC50/LEM3 family / LEM3 (ligand-effect modulator 3) family / CDC50 family / P-type ATPase, subfamily IV / P-type ATPase, C-terminal / P-type ATPase, N-terminal / Phospholipid-translocating ATPase N-terminal / Phospholipid-translocating P-type ATPase C-terminal / 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 / HAD superfamily / HAD-like superfamily
Similarity search - Domain/homology
BERYLLIUM TRIFLUORIDE ION / Chem-IP9 / Chem-POV / Phospholipid-transporting ATPase IC / Cell cycle control protein 50A
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.72 Å
AuthorsDieudonne, T. / Kummerer, F. / Juknaviciute Laursen, M. / Stock, C. / Kock Flygaard, R. / Khalid, S. / Lenoir, G. / Lyons, J.A. / Lindorff-Larsen, K. / Nissen, P.
Funding supportEuropean Union, Denmark, France, United Kingdom, 8items
OrganizationGrant numberCountry
H2020 Marie Curie Actions of the European Commission101024542European Union
LundbeckfondenR310-2018-3713 Denmark
Lundbeckfonden155-2015-2666 Denmark
LundbeckfondenR335-2019-2053 Denmark
Agence Nationale de la Recherche (ANR)ANR-14-CE09-0022 France
French Infrastructure for Integrated Structural Biology (FRISBI)ANR-10-INSB-05 France
Engineering and Physical Sciences Research CouncilEP/X035603 United Kingdom
Engineering and Physical Sciences Research CouncilEP/C030779 United Kingdom
CitationJournal: Nat Commun / Year: 2023
Title: Activation and substrate specificity of the human P4-ATPase ATP8B1.
Authors: Thibaud Dieudonné / Felix Kümmerer / Michelle Juknaviciute Laursen / Charlott Stock / Rasmus Kock Flygaard / Syma Khalid / Guillaume Lenoir / Joseph A Lyons / Kresten Lindorff-Larsen / Poul Nissen /
Abstract: Asymmetric distribution of phospholipids in eukaryotic membranes is essential for cell integrity, signaling pathways, and vesicular trafficking. P4-ATPases, also known as flippases, participate in ...Asymmetric distribution of phospholipids in eukaryotic membranes is essential for cell integrity, signaling pathways, and vesicular trafficking. P4-ATPases, also known as flippases, participate in creating and maintaining this asymmetry through active transport of phospholipids from the exoplasmic to the cytosolic leaflet. Here, we present a total of nine cryo-electron microscopy structures of the human flippase ATP8B1-CDC50A complex at 2.4 to 3.1 Å overall resolution, along with functional and computational studies, addressing the autophosphorylation steps from ATP, substrate recognition and occlusion, as well as a phosphoinositide binding site. We find that the P4-ATPase transport site is occupied by water upon phosphorylation from ATP. Additionally, we identify two different autoinhibited states, a closed and an outward-open conformation. Furthermore, we identify and characterize the PI(3,4,5)P binding site of ATP8B1 in an electropositive pocket between transmembrane segments 5, 7, 8, and 10. Our study also highlights the structural basis of a broad lipid specificity of ATP8B1 and adds phosphatidylinositol as a transport substrate for ATP8B1. We report a critical role of the sn-2 ester bond of glycerophospholipids in substrate recognition by ATP8B1 through conserved S403. These findings provide fundamental insights into ATP8B1 catalytic cycle and regulation, and substrate recognition in P4-ATPases.
History
DepositionMay 1, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 29, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 13, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _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: Phospholipid-transporting ATPase IC
B: Cell cycle control protein 50A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)180,0759
Polymers177,3692
Non-polymers2,7067
Water1629
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area9450 Å2
ΔGint-39 kcal/mol
Surface area59820 Å2

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein Phospholipid-transporting ATPase IC / ATPase class I type 8B member 1 / Familial intrahepatic cholestasis type 1 / P4-ATPase flippase ...ATPase class I type 8B member 1 / Familial intrahepatic cholestasis type 1 / P4-ATPase flippase complex alpha subunit ATP8B1


Mass: 136283.469 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ATP8B1, ATPIC, FIC1, PFIC / Production host: Saccharomyces cerevisiae (brewer's yeast)
References: UniProt: O43520, P-type phospholipid transporter
#2: Protein Cell cycle control protein 50A / P4-ATPase flippase complex beta subunit TMEM30A / Transmembrane protein 30A


Mass: 41085.984 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TMEM30A, C6orf67, CDC50A / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q9NV96

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Sugars , 2 types, 3 molecules

#3: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#8: 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: isolated from a natural source / 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 , 5 types, 13 molecules

#4: Chemical ChemComp-BEF / BERYLLIUM TRIFLUORIDE ION


Mass: 66.007 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: BeF3 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-POV / (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / POPC


Mass: 760.076 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C42H82NO8P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
#7: Chemical ChemComp-IP9 / (2R)-3-{[(R)-{[(1S,2S,3R,4S,5S,6S)-2,6-dihydroxy-3,4,5-tris(phosphonooxy)cyclohexyl]oxy}(hydroxy)phosphoryl]oxy}propane -1,2-diyl dioctanoate / L-a-Phosphatidyl-D-myo-inositol 3,4,5-triphosphate, dioctanoyl / [(2R)-3-[[(2S,3S,5R,6S)-2,6-dihydroxy-3,4,5-triphosphonooxy-cyclohexyl]oxy-hydroxy-phosphoryl]oxy-2-octanoyloxy-propyl] octanoate


Mass: 826.546 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C25H50O22P4 / Feature type: SUBJECT OF INVESTIGATION
#9: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 9 / 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: ATP8B1-CDC50A complex / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.185 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 50 mM MOPS-Tris pH 7, 100 mM KCl, 1 mM DTT, 5 mM MgCl2 supplemented with 0.03 mg.mL-1 LMNG, 0.003 mg.mL-1 CHS, PI(3,4,5)P3 0.0015 mg.mL-1 and 1 mM BeCl3 + 4 mM KF and 0.003 mg.mL-1 POPC
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1900 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.19_4092: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2EPUimage acquisition
4cryoSPARCCTF correction
7Coot0.9model fitting
9PHENIXmodel refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARC3classification
13cryoSPARC33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.72 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 256001 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 7PY4

7py4


Accession code: 7PY4 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00311840
ELECTRON MICROSCOPYf_angle_d0.52616035
ELECTRON MICROSCOPYf_dihedral_angle_d8.1981658
ELECTRON MICROSCOPYf_chiral_restr0.061789
ELECTRON MICROSCOPYf_plane_restr0.0042014

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