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- PDB-7neq: Structure of tariquidar-bound ABCG2 -

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Basic information

Entry
Database: PDB / ID: 7neq
TitleStructure of tariquidar-bound ABCG2
Components
  • 5D3(Fab) heavy chain variable domain
  • 5D3(Fab) light chain variable domain
  • ATP-binding cassette sub-family G member 2
KeywordsMEMBRANE PROTEIN / ABCG2 / tariquidar / substrate / ABC transporter / anticancer drugs / cavity 1 / cryo-EM
Function / homology
Function and homology information


biotin transmembrane transporter activity / biotin transport / riboflavin transport / riboflavin transmembrane transporter activity / renal urate salt excretion / urate metabolic process / urate transmembrane transporter activity / Abacavir transmembrane transport / organic anion transport / external side of apical plasma membrane ...biotin transmembrane transporter activity / biotin transport / riboflavin transport / riboflavin transmembrane transporter activity / renal urate salt excretion / urate metabolic process / urate transmembrane transporter activity / Abacavir transmembrane transport / organic anion transport / external side of apical plasma membrane / organic anion transmembrane transporter activity / xenobiotic transport across blood-brain barrier / export across plasma membrane / ABC-type xenobiotic transporter / Paracetamol ADME / transepithelial transport / Ciprofloxacin ADME / cellular detoxification / ABC-type xenobiotic transporter activity / NFE2L2 regulating MDR associated enzymes / Heme biosynthesis / Heme degradation / lipid transport / xenobiotic transmembrane transporter activity / efflux transmembrane transporter activity / transport across blood-brain barrier / ATPase-coupled transmembrane transporter activity / mitochondrial membrane / brush border membrane / Iron uptake and transport / transmembrane transport / membrane raft / apical plasma membrane / ATP hydrolysis activity / protein homodimerization activity / nucleoplasm / ATP binding / identical protein binding / plasma membrane
Similarity search - Function
ABC transporter family G domain / ABC-2 type transporter / ABC-2 type transporter / ABC-2 type transporter / 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 / tariquidar / Chem-U9N / Broad substrate specificity ATP-binding cassette transporter ABCG2
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.12 Å
AuthorsKowal, J. / Locher, K.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science FoundationTransCure Switzerland
CitationJournal: J Mol Biol / Year: 2021
Title: Structural Basis of Drug Recognition by the Multidrug Transporter ABCG2.
Authors: Julia Kowal / Dongchun Ni / Scott M Jackson / Ioannis Manolaridis / Henning Stahlberg / Kaspar P Locher /
Abstract: ABCG2 is an ATP-binding cassette (ABC) transporter whose function affects the pharmacokinetics of drugs and contributes to multidrug resistance of cancer cells. While its interaction with the ...ABCG2 is an ATP-binding cassette (ABC) transporter whose function affects the pharmacokinetics of drugs and contributes to multidrug resistance of cancer cells. While its interaction with the endogenous substrate estrone-3-sulfate (ES) has been elucidated at a structural level, the recognition and recruitment of exogenous compounds is not understood at sufficiently high resolution. Here we present three cryo-EM structures of nanodisc-reconstituted, human ABCG2 bound to anticancer drugs tariquidar, topotecan and mitoxantrone. To enable structural insight at high resolution, we used Fab fragments of the ABCG2-specific monoclonal antibody 5D3, which binds to the external side of the transporter but does not interfere with drug-induced stimulation of ATPase activity. We observed that the binding pocket of ABCG2 can accommodate a single tariquidar molecule in a C-shaped conformation, similar to one of the two tariquidar molecules bound to ABCB1, where tariquidar acts as an inhibitor. We also found single copies of topotecan and mitoxantrone bound between key phenylalanine residues. Mutagenesis experiments confirmed the functional importance of two residues in the binding pocket, F439 and N436. Using 3D variability analyses, we found a correlation between substrate binding and reduced dynamics of the nucleotide binding domains (NBDs), suggesting a structural explanation for drug-induced ATPase stimulation. Our findings provide additional insight into how ABCG2 differentiates between inhibitors and substrates and may guide a rational design of new modulators and substrates.
History
DepositionFeb 4, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 21, 2021Provider: repository / Type: Initial release
Revision 1.1May 12, 2021Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.title

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Structure visualization

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Assembly

Deposited unit
A: ATP-binding cassette sub-family G member 2
C: 5D3(Fab) light chain variable domain
D: 5D3(Fab) heavy chain variable domain
E: 5D3(Fab) light chain variable domain
F: 5D3(Fab) heavy chain variable domain
B: ATP-binding cassette sub-family G member 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)242,53213
Polymers239,6476
Non-polymers2,8857
Water28816
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy, cryo-EM
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area14190 Å2
ΔGint-70 kcal/mol
Surface area64520 Å2

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Components

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Antibody , 2 types, 4 molecules CEDF

#2: Antibody 5D3(Fab) light chain variable domain


Mass: 23594.016 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)
#3: Antibody 5D3(Fab) heavy chain variable domain


Mass: 23843.633 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)

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Protein / Sugars , 2 types, 4 molecules AB

#1: Protein ATP-binding cassette sub-family G member 2 / Breast cancer resistance protein / CDw338 / Mitoxantrone resistance-associated protein / Placenta- ...Breast cancer resistance protein / CDw338 / Mitoxantrone resistance-associated protein / Placenta-specific ATP-binding cassette transporter / Urate exporter


Mass: 72385.852 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ABCG2, ABCP, BCRP, BCRP1, MXR / Production host: Homo sapiens (human)
References: UniProt: Q9UNQ0, ABC-type xenobiotic transporter
#4: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 4 types, 21 molecules

#5: Chemical ChemComp-U9N / [(2~{S})-3-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-2-decanoyloxy-propyl] octadecanoate


Mass: 635.853 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C33H66NO8P
#6: Chemical ChemComp-CLR / CHOLESTEROL / Cholesterol


Mass: 386.654 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C27H46O
#7: Chemical ChemComp-R1H / tariquidar / ~{N}-[2-[[4-[2-(6,7-dimethoxy-3,4-dihydro-1~{H}-isoquinolin-2-yl)ethyl]phenyl]carbamoyl]-4,5-dimethoxy-phenyl]quinoline-3-carboxamide / Tariquidar


Mass: 646.732 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C38H38N4O6 / Feature type: SUBJECT OF INVESTIGATION / Comment: inhibitor*YM
#8: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 16 / Source method: isolated from a natural source / Formula: H2O

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Details

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
1Multidrug transporter ABCG2 with bound substrate tariquidar and 5D3-Fab variable domainCOMPLEX#1-#30RECOMBINANT
2ATP-binding cassette sub-family G member 2COMPLEX#11RECOMBINANT
35D3(Fab) light- and heavy-chain variable domainsCOMPLEX#2-#31RECOMBINANT
Molecular weightValue: 0.25 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Mus musculus (house mouse)10090
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE

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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 / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 80 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4GctfCTF correction
12RELION3.1classification
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.12 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 166727 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00512800
ELECTRON MICROSCOPYf_angle_d0.96517355
ELECTRON MICROSCOPYf_dihedral_angle_d15.8761800
ELECTRON MICROSCOPYf_chiral_restr0.1021979
ELECTRON MICROSCOPYf_plane_restr0.0042142

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