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- PDB-7niu: Nanodisc reconstituted human ABCB4 in complex with 4B1-Fab and QA... -

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Basic information

Entry
Database: PDB / ID: 7niu
TitleNanodisc reconstituted human ABCB4 in complex with 4B1-Fab and QA2-Fab (apo-inward-open conformation)
Components
  • (4B1 Fab-fragment ...) x 2
  • (QA2 Fab-fragment ...) x 2
  • Isoform 2 of Phosphatidylcholine translocator ABCB4
KeywordsPROTEIN TRANSPORT / ABCB4 / MDR3 / nanodisc / lipid transporter / transporter / phosphatidylcholine
Function / homology
Function and homology information


response to fenofibrate / Defective ABCB4 causes PFIC3, ICP3 and GBD1 / positive regulation of phospholipid transport / positive regulation of cholesterol transport / ABC-type oligopeptide transporter activity / positive regulation of phospholipid translocation / phospholipid transporter activity / bile acid secretion / cellular response to bile acid / phosphatidylcholine floppase activity ...response to fenofibrate / Defective ABCB4 causes PFIC3, ICP3 and GBD1 / positive regulation of phospholipid transport / positive regulation of cholesterol transport / ABC-type oligopeptide transporter activity / positive regulation of phospholipid translocation / phospholipid transporter activity / bile acid secretion / cellular response to bile acid / phosphatidylcholine floppase activity / intercellular canaliculus / P-type phospholipid transporter / phospholipid translocation / clathrin-coated vesicle / lipid homeostasis / ATPase-coupled transmembrane transporter activity / ABC-family proteins mediated transport / transmembrane transport / lipid metabolic process / PPARA activates gene expression / membrane raft / apical plasma membrane / focal adhesion / ATP hydrolysis activity / extracellular exosome / nucleoplasm / ATP binding / membrane / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Type 1 protein exporter / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. ...Type 1 protein exporter / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
CHOLESTEROL / Phosphatidylcholine translocator ABCB4
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsNosol, K. / Locher, K.P.
Funding support Switzerland, United States, 3items
OrganizationGrant numberCountry
Swiss National Science Foundation310030_189111 Switzerland
Swiss National Science Foundation310030_155563 Switzerland
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM117372 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2021
Title: Structures of ABCB4 provide insight into phosphatidylcholine translocation.
Authors: Kamil Nosol / Rose Bang-Sørensen / Rossitza N Irobalieva / Satchal K Erramilli / Bruno Stieger / Anthony A Kossiakoff / Kaspar P Locher /
Abstract: ABCB4 is expressed in hepatocytes and translocates phosphatidylcholine into bile canaliculi. The mechanism of specific lipid recruitment from the canalicular membrane, which is essential to mitigate ...ABCB4 is expressed in hepatocytes and translocates phosphatidylcholine into bile canaliculi. The mechanism of specific lipid recruitment from the canalicular membrane, which is essential to mitigate the cytotoxicity of bile salts, is poorly understood. We present cryogenic electron microscopy structures of human ABCB4 in three distinct functional conformations. An apo-inward structure reveals how phospholipid can be recruited from the inner leaflet of the membrane without flipping its orientation. An occluded structure reveals a single phospholipid molecule in a central cavity. Its choline moiety is stabilized by cation-π interactions with an essential tryptophan residue, rationalizing the specificity of ABCB4 for phosphatidylcholine. In an inhibitor-bound structure, a posaconazole molecule blocks phospholipids from reaching the central cavity. Using a proteoliposome-based translocation assay with fluorescently labeled phosphatidylcholine analogs, we recapitulated the substrate specificity of ABCB4 in vitro and confirmed the role of the key tryptophan residue. Our results provide a structural basis for understanding an essential translocation step in the generation of bile and its sensitivity to azole drugs.
History
DepositionFeb 14, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 25, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Isoform 2 of Phosphatidylcholine translocator ABCB4
D: QA2 Fab-fragment light chain
E: QA2 Fab-fragment heavy chain
B: 4B1 Fab-fragment light chain
C: 4B1 Fab-fragment heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)238,7877
Polymers238,0135
Non-polymers7732
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area11540 Å2
ΔGint-59 kcal/mol
Surface area88550 Å2

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Components

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Antibody , 4 types, 4 molecules DEBC

#2: Antibody QA2 Fab-fragment light chain


Mass: 23258.783 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#3: Antibody QA2 Fab-fragment heavy chain


Mass: 24817.670 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#4: Antibody 4B1 Fab-fragment light chain


Mass: 23346.957 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#5: Antibody 4B1 Fab-fragment heavy chain


Mass: 25754.572 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)

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Protein / Non-polymers , 2 types, 3 molecules A

#1: Protein Isoform 2 of Phosphatidylcholine translocator ABCB4 / ATP-binding cassette sub-family B member 4 / Multidrug resistance protein 3 / P-glycoprotein 3


Mass: 140835.375 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ABCB4, MDR3, PGY3 / Production host: Homo sapiens (human)
References: UniProt: P21439, P-type phospholipid transporter
#6: Chemical ChemComp-CLR / CHOLESTEROL / Cholesterol


Mass: 386.654 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H46O

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Details

Has ligand of interestN

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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

Component
IDNameTypeEntity IDParent-IDSource
1Nanodisc reconstituted human ABCB4 in complex with 4B1-Fab and QA2-Fab (apo-inward-open conformation)COMPLEX#1-#50MULTIPLE SOURCES
2phosphatidylcholine translocator ABCB4COMPLEX#11RECOMBINANT
3QA2 Fab-fragment: light and heavy chainCOMPLEX#2-#31RECOMBINANT
44B1 Fab-fragment: light and heavy chainCOMPLEX#4-#51RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.34 MDaYES
14
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Homo sapiens (human)9606
44Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Escherichia coli (E. coli)562
44Escherichia coli (E. coli)562
Buffer solutionpH: 7.4
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 FIELDBright-field microscopy / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 80.4 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 34430 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00815772
ELECTRON MICROSCOPYf_angle_d0.88721384
ELECTRON MICROSCOPYf_dihedral_angle_d16.2222176
ELECTRON MICROSCOPYf_chiral_restr0.0522439
ELECTRON MICROSCOPYf_plane_restr0.0062718

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