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- EMDB-11671: Nanodisc reconstituted human ABCB1 in complex with MRK16 Fab and ... -

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Basic information

Entry
Database: EMDB / ID: EMD-11671
TitleNanodisc reconstituted human ABCB1 in complex with MRK16 Fab and tariquidar
Map datatariquidar-bound ABCB1-MRK16-Fab
Sample
  • Complex: Nanodisc reconstituted human ABCB1 in complex with MRK16 Fab and tariquidar
    • Complex: Multidrug resistance protein 1P-glycoprotein
      • Protein or peptide: Multidrug resistance protein 1P-glycoprotein
    • Complex: MRK16 Fab-fragment light and heavy chain
      • Protein or peptide: MRK16 Fab-fragment light chain
      • Protein or peptide: MRK16 Fab-fragment heavy chain
  • Ligand: tariquidar
  • Ligand: CHOLESTEROL
Function / homology
Function and homology information


positive regulation of anion channel activity / ceramide translocation / terpenoid transport / ceramide floppase activity / carboxylic acid transmembrane transport / carboxylic acid transmembrane transporter activity / floppase activity / Abacavir transmembrane transport / external side of apical plasma membrane / regulation of response to osmotic stress ...positive regulation of anion channel activity / ceramide translocation / terpenoid transport / ceramide floppase activity / carboxylic acid transmembrane transport / carboxylic acid transmembrane transporter activity / floppase activity / Abacavir transmembrane transport / external side of apical plasma membrane / regulation of response to osmotic stress / Atorvastatin ADME / phosphatidylcholine floppase activity / phosphatidylethanolamine flippase activity / xenobiotic transport across blood-brain barrier / xenobiotic detoxification by transmembrane export across the plasma membrane / export across plasma membrane / ABC-type xenobiotic transporter / transepithelial transport / P-type phospholipid transporter / ABC-type xenobiotic transporter activity / phospholipid translocation / Prednisone ADME / xenobiotic transmembrane transporter activity / efflux transmembrane transporter activity / transmembrane transporter activity / transport across blood-brain barrier / ATPase-coupled transmembrane transporter activity / xenobiotic metabolic process / regulation of chloride transport / stem cell proliferation / ABC-family proteins mediated transport / transmembrane transport / G2/M transition of mitotic cell cycle / response to xenobiotic stimulus / apical plasma membrane / ubiquitin protein ligase binding / cell surface / ATP hydrolysis activity / extracellular exosome / ATP binding / membrane / plasma membrane / 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
ATP-dependent translocase ABCB1
Similarity search - Component
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsNosol K / Locher KP
Funding support Switzerland, 1 items
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.
History
DepositionAug 25, 2020-
Header (metadata) releaseOct 14, 2020-
Map releaseOct 14, 2020-
UpdateOct 28, 2020-
Current statusOct 28, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.011
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.011
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7a6e
  • Surface level: 0.011
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7a6e
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11671.png

Downloads & links

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Map

FileDownload / File: emd_11671.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationtariquidar-bound ABCB1-MRK16-Fab
Voxel sizeX=Y=Z: 0.84 Å
Density
Contour LevelBy AUTHOR: 0.013 / Movie #1: 0.011
Minimum - Maximum-0.036109023 - 0.064618334
Average (Standard dev.)-0.00000789 (±0.00139182)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 322.56 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.840.840.84
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z322.560322.560322.560
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ161186271
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.0360.065-0.000

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Supplemental data

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Sample components

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Entire : Nanodisc reconstituted human ABCB1 in complex with MRK16 Fab and ...

EntireName: Nanodisc reconstituted human ABCB1 in complex with MRK16 Fab and tariquidar
Components
  • Complex: Nanodisc reconstituted human ABCB1 in complex with MRK16 Fab and tariquidar
    • Complex: Multidrug resistance protein 1P-glycoprotein
      • Protein or peptide: Multidrug resistance protein 1P-glycoprotein
    • Complex: MRK16 Fab-fragment light and heavy chain
      • Protein or peptide: MRK16 Fab-fragment light chain
      • Protein or peptide: MRK16 Fab-fragment heavy chain
  • Ligand: tariquidar
  • Ligand: CHOLESTEROL

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Supramolecule #1: Nanodisc reconstituted human ABCB1 in complex with MRK16 Fab and ...

SupramoleculeName: Nanodisc reconstituted human ABCB1 in complex with MRK16 Fab and tariquidar
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Molecular weightExperimental: 240 KDa

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Supramolecule #2: Multidrug resistance protein 1

SupramoleculeName: Multidrug resistance protein 1 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)

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Supramolecule #3: MRK16 Fab-fragment light and heavy chain

SupramoleculeName: MRK16 Fab-fragment light and heavy chain / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Homo sapiens (human)

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Macromolecule #1: Multidrug resistance protein 1

MacromoleculeName: Multidrug resistance protein 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: ABC-type xenobiotic transporter
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 141.628781 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDLEGDRNGG AKKKNFFKLN NKSEKDKKEK KPTVSVFSMF RYSNWLDKLY MVVGTLAAII HGAGLPLMML VFGEMTDIFA NAGNLEDLM SNITNRSDIN DTGFFMNLEE DMTRYAYYYS GIGAGVLVAA YIQVSFWCLA AGRQIHKIRK QFFHAIMRQE I GWFDVHDV ...String:
MDLEGDRNGG AKKKNFFKLN NKSEKDKKEK KPTVSVFSMF RYSNWLDKLY MVVGTLAAII HGAGLPLMML VFGEMTDIFA NAGNLEDLM SNITNRSDIN DTGFFMNLEE DMTRYAYYYS GIGAGVLVAA YIQVSFWCLA AGRQIHKIRK QFFHAIMRQE I GWFDVHDV GELNTRLTDD VSKINEGIGD KIGMFFQSMA TFFTGFIVGF TRGWKLTLVI LAISPVLGLS AAVWAKILSS FT DKELLAY AKAGAVAEEV LAAIRTVIAF GGQKKELERY NKNLEEAKRI GIKKAITANI SIGAAFLLIY ASYALAFWYG TTL VLSGEY SIGQVLTVFF SVLIGAFSVG QASPSIEAFA NARGAAYEIF KIIDNKPSID SYSKSGHKPD NIKGNLEFRN VHFS YPSRK EVKILKGLNL KVQSGQTVAL VGNSGCGKST TVQLMQRLYD PTEGMVSVDG QDIRTINVRF LREIIGVVSQ EPVLF ATTI AENIRYGREN VTMDEIEKAV KEANAYDFIM KLPHKFDTLV GERGAQLSGG QKQRIAIARA LVRNPKILLL DEATSA LDT ESEAVVQVAL DKARKGRTTI VIAHRLSTVR NADVIAGFDD GVIVEKGNHD ELMKEKGIYF KLVTMQTAGN EVELENA AD ESKSEIDALE MSSNDSRSSL IRKRSTRRSV RGSQAQDRKL STKEALDESI PPVSFWRIMK LNLTEWPYFV VGVFCAII N GGLQPAFAII FSKIIGVFTR IDDPETKRQN SNLFSLLFLA LGIISFITFF LQGFTFGKAG EILTKRLRYM VFRSMLRQD VSWFDDPKNT TGALTTRLAN DAAQVKGAIG SRLAVITQNI ANLGTGIIIS FIYGWQLTLL LLAIVPIIAI AGVVEMKMLS GQALKDKKE LEGAGKIATE AIENFRTVVS LTQEQKFEHM YAQSLQVPYR NSLRKAHIFG ITFSFTQAMM YFSYAGCFRF G AYLVAHKL MSFEDVLLVF SAVVFGAMAV GQVSSFAPDY AKAKISAAHI IMIIEKTPLI DSYSTEGLMP NTLEGNVTFG EV VFNYPTR PDIPVLQGLS LEVKKGQTLA LVGSSGCGKS TVVQLLERFY DPLAGKVLLD GKEIKRLNVQ WLRAHLGIVS QEP ILFDCS IAENIAYGDN SRVVSQEEIV RAAKEANIHA FIESLPNKYS TKVGDKGTQL SGGQKQRIAI ARALVRQPHI LLLD EATSA LDTESEKVVQ EALDKAREGR TCIVIAHRLS TIQNADLIVV FQNGRVKEHG THQQLLAQKG IYFSMVSVQA GTKRQ

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Macromolecule #2: MRK16 Fab-fragment light chain

MacromoleculeName: MRK16 Fab-fragment light chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 24.139758 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DVLMTQTPVS LSVSLGDQAS ISCRSSQSIV HSTGNTYLEW YLQKPGQSPK LLIYKISNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLG VYYCFQASHA PRTFGGGTKL EIKRADAAPT VSIFPPSSEQ LTSGGASVVC FLNNFYPKDI NVKWKIDGSE R QNGVLNSW ...String:
DVLMTQTPVS LSVSLGDQAS ISCRSSQSIV HSTGNTYLEW YLQKPGQSPK LLIYKISNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLG VYYCFQASHA PRTFGGGTKL EIKRADAAPT VSIFPPSSEQ LTSGGASVVC FLNNFYPKDI NVKWKIDGSE R QNGVLNSW TDQDSKDSTY SMSSTLTLTK DEYERHNSYT CEATHKTSTS PIVKSFNRNE C

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Macromolecule #3: MRK16 Fab-fragment heavy chain

MacromoleculeName: MRK16 Fab-fragment heavy chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.415236 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: EVILVESGGG LVKPGGSLKL SCAASGFTFS SYTMSWVRQT PEKRLEWVAT ISSGGGNTYY PDSVKGRFTI SRDNAKNNLY LQMSSLRSE DTALYYCARY YRYEAWFASW GQGTLVTVSA AKTTAPSVYP LAPVCGDTTG SSVTLGCLVK GYFPEPVTLT W NSGSLSSG ...String:
EVILVESGGG LVKPGGSLKL SCAASGFTFS SYTMSWVRQT PEKRLEWVAT ISSGGGNTYY PDSVKGRFTI SRDNAKNNLY LQMSSLRSE DTALYYCARY YRYEAWFASW GQGTLVTVSA AKTTAPSVYP LAPVCGDTTG SSVTLGCLVK GYFPEPVTLT W NSGSLSSG VHTFPAVLQS DLYTLSSSVT VTSSTWPSQS ITCNVAHPAS STKVDKKIEP

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Macromolecule #4: tariquidar

MacromoleculeName: tariquidar / type: ligand / ID: 4 / Number of copies: 2 / Formula: R1H
Molecular weightTheoretical: 646.732 Da
Chemical component information

ChemComp-R1H:
tariquidar / inhibitor*YM / Tariquidar

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Macromolecule #5: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 5 / Number of copies: 11 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL / Cholesterol

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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

Concentration0.4 mg/mL
BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 32.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 160295
FSC plot (resolution estimation)

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