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- EMDB-17261: Cryo-EM structure of ATP8B1-CDC50A in E2P active conformation wit... -

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Entry
Database: EMDB / ID: EMD-17261
TitleCryo-EM structure of ATP8B1-CDC50A in E2P active conformation with bound PC
Map datasharpened map by PHENIX autosharpen B factor applied 60
Sample
  • Complex: ATP8B1-CDC50A complex
    • Protein or peptide: Phospholipid-transporting ATPase IC
    • Protein or peptide: Cell cycle control protein 50A
  • Ligand: BERYLLIUM TRIFLUORIDE ION
  • Ligand: MAGNESIUM ION
  • Ligand: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
  • Ligand: (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
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: water
Keywordslipid transporter autoinhibition P-type ATPase P4-ATPase CDC50A / MEMBRANE PROTEIN
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
Phospholipid-transporting ATPase IC / Cell cycle control protein 50A
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle 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 JA / Lindorff-Larsen K / Nissen P
Funding supportEuropean Union, Denmark, France, United Kingdom, 8 items
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, 2023-
Header (metadata) releaseNov 29, 2023-
Map releaseNov 29, 2023-
UpdateNov 29, 2023-
Current statusNov 29, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17261.png

Downloads & links

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Map

FileDownload / File: emd_17261.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map by PHENIX autosharpen B factor applied 60
Projections & slices

Image control

Size
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AxesX (Sec.)Y (Row.)Z (Col.)
1.04 Å/pix.
x 240 pix.
= 248.448 Å		1.04 Å/pix.
x 240 pix.
= 248.448 Å		1.04 Å/pix.
x 240 pix.
= 248.448 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0352 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.0
Minimum - Maximum-30.594245999999998 - 49.582253000000001
Average (Standard dev.)-0.000000000002202 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 248.448 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: raw map before sharpening

Fileemd_17261_additional_1.map
Annotationraw map before sharpening
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram (log scale)

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Half map: half map A

Fileemd_17261_half_map_1.map
Annotationhalf map A
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B

Fileemd_17261_half_map_2.map
Annotationhalf map B
Projections & Slices
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Sample components

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Entire : ATP8B1-CDC50A complex

EntireName: ATP8B1-CDC50A complex
Components
  • Complex: ATP8B1-CDC50A complex
    • Protein or peptide: Phospholipid-transporting ATPase IC
    • Protein or peptide: Cell cycle control protein 50A
  • Ligand: BERYLLIUM TRIFLUORIDE ION
  • Ligand: MAGNESIUM ION
  • Ligand: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
  • Ligand: (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
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: water

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Supramolecule #1: ATP8B1-CDC50A complex

SupramoleculeName: ATP8B1-CDC50A complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 185 KDa

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Macromolecule #1: Phospholipid-transporting ATPase IC

MacromoleculeName: Phospholipid-transporting ATPase IC / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: P-type phospholipid transporter
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 136.283469 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MSTERDSETT FDEDSQPNDE VVPYSDDETE DELDDQGSAV EPEQNRVNRE AEENREPFRK ECTWQVKAND RKYHEQPHFM NTKFLCIKE SKYANNAIKT YKYNAFTFIP MNLFEQFKRA ANLYFLALLI LQAVPQISTL AWYTTLVPLL VVLGVTAIKD L VDDVARHK ...String:
MSTERDSETT FDEDSQPNDE VVPYSDDETE DELDDQGSAV EPEQNRVNRE AEENREPFRK ECTWQVKAND RKYHEQPHFM NTKFLCIKE SKYANNAIKT YKYNAFTFIP MNLFEQFKRA ANLYFLALLI LQAVPQISTL AWYTTLVPLL VVLGVTAIKD L VDDVARHK MDKEINNRTC EVIKDGRFKV AKWKEIQVGD VIRLKKNDFV PADILLLSSS EPNSLCYVET AELDGETNLK FK MSLEITD QYLQREDTLA TFDGFIECEE PNNRLDKFTG TLFWRNTSFP LDADKILLRG CVIRNTDFCH GLVIFAGADT KIM KNSGKT RFKRTKIDYL MNYMVYTIFV VLILLSAGLA IGHAYWEAQV GNSSWYLYDG EDDTPSYRGF LIFWGYIIVL NTMV PISLY VSVEVIRLGQ SHFINWDLQM YYAEKDTPAK ARTTTLNEQL GQIHYIFSDK TGTLTQNIMT FKKCCINGQI YGDHR DASQ HNHNKIEQVD FSWNTYADGK LAFYDHYLIE QIQSGKEPEV RQFFFLLAVC HTVMVDRTDG QLNYQAASPD EGALVN AAR NFGFAFLART QNTITISELG TERTYNVLAI LDFNSDRKRM SIIVRTPEGN IKLYCKGADT VIYERLHRMN PTKQETQ DA LDIFANETLR TLCLCYKEIE EKEFTEWNKK FMAASVASTN RDEALDKVYE EIEKDLILLG ATAIEDKLQD GVPETISK L AKADIKIWVL TGDKKETAEN IGFACELLTE DTTICYGEDI NSLLHARMEN QRNRGGVYAK FAPPVQESFF PPGGNRALI ITGSWLNEIL LEKKTKRNKI LKLKFPRTEE ERRMRTQSKR RLEAKKEQRQ KNFVDLACEC SAVICCRVTP KQKAMVVDLV KRYKKAITL AIGDGANDVN MIKTAHIGVG ISGQEGMQAV MSSDYSFAQF RYLQRLLLVH GRWSYIRMCK FLRYFFYKNF A FTLVHFWY SFFNGYSAQT AYEDWFITLY NVLYTSLPVL LMGLLDQDVS DKLSLRFPGL YIVGQRDLLF NYKRFFVSLL HG VLTSMIL FFIPLGAYLQ TVGQDGEAPS DYQSFAVTIA SALVITVNFQ IGLDTSYWTF VNAFSIFGSI ALYFGIMFDF HSA GIHVLF PSAFQFTGTA SNALRQPYIW LTIILTVAVC LLPVVAIRFL SMTIWPSESD KIQKHRKRL

UniProtKB: Phospholipid-transporting ATPase IC

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Macromolecule #2: Cell cycle control protein 50A

MacromoleculeName: Cell cycle control protein 50A / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 41.085984 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MLGGMAMNYN AKDEVDGGPP CAPGGTAKTR RPDNTAFKQQ RLPAWQPILT AGTVLPIFFI IGLIFIPIGI GIFVTSNNIR EIEIDYTGT EPSSPCNKCL SPDVTPCFCT INFTLEKSFE GNVFMYYGLS NFYQNHRRYV KSRDDSQLNG DSSALLNPSK E CEPYRRNE ...String:
MLGGMAMNYN AKDEVDGGPP CAPGGTAKTR RPDNTAFKQQ RLPAWQPILT AGTVLPIFFI IGLIFIPIGI GIFVTSNNIR EIEIDYTGT EPSSPCNKCL SPDVTPCFCT INFTLEKSFE GNVFMYYGLS NFYQNHRRYV KSRDDSQLNG DSSALLNPSK E CEPYRRNE DKPIAPCGAI ANSMFNDTLE LFLIGNDSYP IPIALKKKGI AWWTDKNVKF RNPPGGDNLE ERFKGTTKPV NW LKPVYML DSDPDNNGFI NEDFIVWMRT AALPTFRKLY RLIERKSDLH PTLPAGRYSL NVTYNYPVHY FDGRKRMILS TIS WMGGKN PFLGIAYIAV GSISFLLGVV LLVINHKYRN SSNTADITI

UniProtKB: Cell cycle control protein 50A

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Macromolecule #4: BERYLLIUM TRIFLUORIDE ION

MacromoleculeName: BERYLLIUM TRIFLUORIDE ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: BEF
Molecular weightTheoretical: 66.007 Da
Chemical component information

ChemComp-BEF:
BERYLLIUM TRIFLUORIDE ION

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(tri...

MacromoleculeName: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
type: ligand / ID: 6 / Number of copies: 1 / Formula: POV
Molecular weightTheoretical: 760.076 Da
Chemical component information

ChemComp-POV:
(2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / phospholipid*YM

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Macromolecule #7: (2R)-3-{[(R)-{[(1S,2S,3R,4S,5S,6S)-2,6-dihydroxy-3,4,5-tris(phosp...

MacromoleculeName: (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
type: ligand / ID: 7 / Number of copies: 1 / Formula: IP9
Molecular weightTheoretical: 826.546 Da

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Macromolecule #8: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 8 / 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 #9: water

MacromoleculeName: water / type: ligand / ID: 9 / Number of copies: 9 / 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

Concentration5 mg/mL
BufferpH: 7
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details50 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

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
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 / Cs: 2.7 mm / Nominal defocus max: 1.9000000000000001 µm / Nominal defocus min: 0.7000000000000001 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.72 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3) / Number images used: 256001
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7py4


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8ox9:
Cryo-EM structure of ATP8B1-CDC50A in E2P active conformation with bound PC

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