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- PDB-6q81: Structure of P-glycoprotein(ABCB1) in the post-hydrolytic state -

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Basic information

Entry
Database: PDB / ID: 6q81
TitleStructure of P-glycoprotein(ABCB1) in the post-hydrolytic state
ComponentsP-glycoprotein (ABCB1)
KeywordsMEMBRANE PROTEIN / P-glycoprotein / ABCB1 / ATP-binding cassette / transporter / membrane protein / protein structure
Function / homologyABC transporter, conserved site / ABC transporter / ABC transporter-like / AAA+ ATPase domain / ABC transporter type 1, transmembrane domain / ABC transporter type 1, transmembrane domain superfamily / Type I protein exporter / P-loop containing nucleoside triphosphate hydrolase / ABC transporter transmembrane region / ABC transporters family signature. ...ABC transporter, conserved site / ABC transporter / ABC transporter-like / AAA+ ATPase domain / ABC transporter type 1, transmembrane domain / ABC transporter type 1, transmembrane domain superfamily / Type I protein exporter / P-loop containing nucleoside triphosphate hydrolase / ABC transporter transmembrane region / ABC transporters family signature. / ATP-binding cassette, ABC transporter-type domain profile. / ABC transporter integral membrane type-1 fused domain profile. / ABC-family proteins mediated transport / hormone transport / establishment of blood-brain barrier / carbohydrate export / establishment of blood-retinal barrier / drug transport across blood-brain barrier / regulation of intestinal absorption / daunorubicin transport / positive regulation of response to drug / drug transport / positive regulation of establishment of Sertoli cell barrier / ceramide translocation / phosphatidylethanolamine-translocating ATPase activity / positive regulation of anion channel activity / ceramide-translocating ATPase activity / ABC-type xenobiotic transporter / phosphatidylcholine-translocating ATPase activity / protein localization to bicellular tight junction / phospholipid translocation / regulation of response to osmotic stress / drug export / xenobiotic transmembrane transporting ATPase activity / stem cell proliferation / maintenance of permeability of blood-brain barrier / intercellular canaliculus / intestinal absorption / drug transmembrane transport / brush border membrane / negative regulation of cell death / ATPase activity, coupled / regulation of chloride transport / apical part of cell / apical plasma membrane / G2/M transition of mitotic cell cycle / cell surface / membrane / integral component of membrane / ATP binding / plasma membrane / Multidrug resistance protein 1A
Function and homology information
Specimen sourceMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 7.9 Å resolution
AuthorsFord, R.C. / Thonghin, N. / Collins, R.F. / Barbieri, A. / Shafi, T. / Siebert, A.
CitationJournal: BMC Struct. Biol. / Year: 2018
Title: Novel features in the structure of P-glycoprotein (ABCB1) in the post-hydrolytic state as determined at 7.9 Å resolution.
Authors: Nopnithi Thonghin / Richard F Collins / Alessandro Barbieri / Talha Shafi / Alistair Siebert / Robert C Ford
Abstract: BACKGROUND: P-glycoprotein (ABCB1) is an ATP-binding cassette transporter that plays an important role in the clearance of drugs and xenobiotics and is associated with multi-drug resistance in cancer. ...BACKGROUND: P-glycoprotein (ABCB1) is an ATP-binding cassette transporter that plays an important role in the clearance of drugs and xenobiotics and is associated with multi-drug resistance in cancer. Although several P-glycoprotein structures are available, these are either at low resolution, or represent mutated and/or quiescent states of the protein.
RESULTS: In the post-hydrolytic state the structure of the wild-type protein has been resolved at about 8 Å resolution. The cytosolic nucleotide-binding domains (NBDs) are separated but ADP remains bound, especially at the first NBD. Gaps in the transmembrane domains (TMDs) that connect to an inner hydrophilic cavity are filled by density emerging from the annular detergent micelle. The NBD-TMD linker is partly resolved, being located between the NBDs and close to the Signature regions involved in cooperative NBD dimerization. This, and the gap-filling detergent suggest steric impediment to NBD dimerization in the post-hydrolytic state. Two central regions of density lie in two predicted drug-binding sites, implying that the protein may adventitiously bind hydrophobic substances even in the post-hydrolytic state. The previously unresolved N-terminal extension was observed, and the data suggests these 30 residues interact with the headgroup region of the lipid bilayer.
CONCLUSION: The structural data imply that (i) a low basal ATPase activity is ensured by steric blockers of NBD dimerization and (ii) allocrite access to the central cavity may be structurally linked to NBD dimerization, giving insights into the mechanism of drug-stimulation of P-glycoprotein activity.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Dec 13, 2018 / Release: Dec 26, 2018

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Assembly

Deposited unit
A: P-glycoprotein (ABCB1)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)141,6613
Polyers140,8071
Non-polymers8542
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)1100
ΔGint (kcal/M)-12
Surface area (Å2)55040
MethodPISA

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Components

#1: Protein/peptide P-glycoprotein (ABCB1)


Mass: 140806.766 Da / Num. of mol.: 1 / Source: (gene. exp.) Mus musculus (house mouse) / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P21447
#2: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 2 / Formula: C10H15N5O10P2 / Adenosine diphosphate / Comment: ADP (energy-carrying molecule) *YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: P-glycoprotein trapped in the post-hydrolytic state. / Type: ORGANELLE OR CELLULAR COMPONENT / Details: ATP and Vanadate added / Entity ID: 1 / Source: RECOMBINANT
Molecular weightValue: 142 kDa/nm / Experimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Saccharomyces cerevisiae (baker's yeast)
Buffer solutionpH: 8
SpecimenEmbedding 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
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 70 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameCategory
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
SymmetryPoint symmetry: C1
3D reconstructionMethod: SINGLE PARTICLESingle particle analysis / Resolution: 7.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 135357 / Symmetry type: POINT

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