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- PDB-7a6f: Nanodisc reconstituted human ABCB1 in complex with MRK16 Fab and ... -

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Basic information

Entry
Database: PDB / ID: 7a6f
TitleNanodisc reconstituted human ABCB1 in complex with MRK16 Fab and zosuquidar
Components
  • MRK16 Fab-fragment heavy chain
  • MRK16 Fab-fragment light chain
  • Multidrug resistance protein 1Multiple drug resistance
KeywordsTRANSPORT PROTEIN / P-glycoprotein / MDR1 / nanodisc
Function / homology
Function and homology information


ceramide floppase activity / ceramide translocation / positive regulation of anion channel activity / floppase activity / external side of apical plasma membrane / phosphatidylcholine floppase activity / transporter activity / phosphatidylethanolamine flippase activity / regulation of response to osmotic stress / xenobiotic transport across blood-brain barrier ...ceramide floppase activity / ceramide translocation / positive regulation of anion channel activity / floppase activity / external side of apical plasma membrane / phosphatidylcholine floppase activity / transporter activity / phosphatidylethanolamine flippase activity / regulation of response to osmotic stress / xenobiotic transport across blood-brain barrier / ABC-type xenobiotic transporter / P-type phospholipid transporter / export across plasma membrane / stem cell proliferation / transepithelial transport / ATPase-coupled xenobiotic transmembrane transporter activity / phospholipid translocation / xenobiotic transmembrane transporter activity / transport across blood-brain barrier / efflux transmembrane transporter activity / ATPase-coupled transmembrane transporter activity / regulation of chloride transport / transmembrane transport / G2/M transition of mitotic cell cycle / apical plasma membrane / response to drug / ATPase activity / ubiquitin protein ligase binding / cell surface / extracellular exosome / membrane / integral component of membrane / ATP binding / plasma membrane
AAA+ ATPase domain / Type I protein exporter / ABC transporter type 1, transmembrane domain superfamily / Multidrug resistance protein 1 / P-loop containing nucleoside triphosphate hydrolase / ABC transporter, conserved site / ABC transporter type 1, transmembrane domain / ABC transporter-like
ATP-dependent translocase ABCB1
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsNosol, K. / Locher, K.P.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science Foundation310030_189111 Switzerland
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: Cryo-EM structures reveal distinct mechanisms of inhibition of the human multidrug transporter ABCB1.
Authors: Kamil Nosol / Ksenija Romane / Rossitza N Irobalieva / Amer Alam / Julia Kowal / Naoya Fujita / Kaspar P Locher /
Abstract: ABCB1 detoxifies cells by exporting diverse xenobiotic compounds, thereby limiting drug disposition and contributing to multidrug resistance in cancer cells. Multiple small-molecule inhibitors and ...ABCB1 detoxifies cells by exporting diverse xenobiotic compounds, thereby limiting drug disposition and contributing to multidrug resistance in cancer cells. Multiple small-molecule inhibitors and inhibitory antibodies have been developed for therapeutic applications, but the structural basis of their activity is insufficiently understood. We determined cryo-EM structures of nanodisc-reconstituted, human ABCB1 in complex with the Fab fragment of the inhibitory, monoclonal antibody MRK16 and bound to a substrate (the antitumor drug vincristine) or to the potent inhibitors elacridar, tariquidar, or zosuquidar. We found that inhibitors bound in pairs, with one molecule lodged in the central drug-binding pocket and a second extending into a phenylalanine-rich cavity that we termed the "access tunnel." This finding explains how inhibitors can act as substrates at low concentration, but interfere with the early steps of the peristaltic extrusion mechanism at higher concentration. Our structural data will also help the development of more potent and selective ABCB1 inhibitors.
Validation Report
SummaryFull reportAbout validation report
History
DepositionAug 25, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 14, 2020Provider: repository / Type: Initial release

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Assembly

Deposited unit
A: Multidrug resistance protein 1
B: MRK16 Fab-fragment light chain
C: MRK16 Fab-fragment heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)193,33213
Polymers189,1843
Non-polymers4,14810
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area10020 Å2
ΔGint-23 kcal/mol
Surface area71700 Å2
MethodPISA

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Components

#1: Protein Multidrug resistance protein 1 / Multiple drug resistance / ATP-binding cassette sub-family B member 1 / P-glycoprotein 1


Mass: 141628.781 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ABCB1, MDR1, PGY1 / Production host: Homo sapiens (human)
References: UniProt: P08183, ABC-type xenobiotic transporter, P-type phospholipid transporter
#2: Antibody MRK16 Fab-fragment light chain


Mass: 24139.758 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Homo sapiens (human)
#3: Antibody MRK16 Fab-fragment heavy chain


Mass: 23415.236 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Homo sapiens (human)
#4: Chemical ChemComp-ZQU / Zosuquidar / Zosuquidar


Mass: 527.604 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C32H31F2N3O2 / Feature type: SUBJECT OF INVESTIGATION / Comment: antineoplastic*YM
#5: Chemical
ChemComp-CLR / CHOLESTEROL / Cholesterol


Mass: 386.654 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C27H46O
Has ligand of interestY

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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 ABCB1 in complex with MRK16 Fab and zosuquidarCOMPLEX#1-#30MULTIPLE SOURCES
2Multidrug resistance protein 1Multiple drug resistanceCOMPLEX#11RECOMBINANT
3MRK16 Fab-fragment light and heavy chainCOMPLEX#2-#31RECOMBINANT
Molecular weightValue: 0.24 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Homo sapiens (human)9606
Buffer solutionpH: 7.4
SpecimenConc.: 0.4 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 / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 32 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: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 166803 / Symmetry type: POINT
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00712934
ELECTRON MICROSCOPYf_angle_d0.92317564
ELECTRON MICROSCOPYf_dihedral_angle_d22.731802
ELECTRON MICROSCOPYf_chiral_restr0.172030
ELECTRON MICROSCOPYf_plane_restr0.0052159

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