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- EMDB-12290: Structure of tariquidar-bound ABCG2 -

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Basic information

Entry
Database: EMDB / ID: EMD-12290
TitleStructure of tariquidar-bound ABCG2
Map dataHuman ABCG2 with tariquidar substrate map
Sample
  • Complex: Multidrug transporter ABCG2 with bound substrate tariquidar and 5D3-Fab variable domain
    • Complex: ATP-binding cassette sub-family G member 2
      • Protein or peptide: ATP-binding cassette sub-family G member 2
    • Complex: 5D3(Fab) light- and heavy-chain variable domains
      • Protein or peptide: 5D3(Fab) light chain variable domain
      • Protein or peptide: 5D3(Fab) heavy chain variable domain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: [(2~{S})-3-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-2-decanoyloxy-propyl] octadecanoate
  • Ligand: CHOLESTEROL
  • Ligand: tariquidar
  • Ligand: water
KeywordsABCG2 / tariquidar / substrate / ABC transporter / anticancer drugs / cavity 1 / cryo-EM / MEMBRANE PROTEIN
Function / homology
Function and homology information


biotin transmembrane transporter activity / biotin transport / riboflavin transport / riboflavin transmembrane transporter activity / sphingolipid transporter activity / renal urate salt excretion / Abacavir transmembrane transport / urate metabolic process / urate transmembrane transporter activity / sphingolipid biosynthetic process ...biotin transmembrane transporter activity / biotin transport / riboflavin transport / riboflavin transmembrane transporter activity / sphingolipid transporter activity / renal urate salt excretion / Abacavir transmembrane transport / urate metabolic process / urate transmembrane transporter activity / sphingolipid biosynthetic process / external side of apical plasma membrane / Sphingolipid de novo biosynthesis / organic anion transport / xenobiotic transport across blood-brain barrier / organic anion transmembrane transporter activity / transepithelial transport / export across plasma membrane / Ciprofloxacin ADME / Paracetamol ADME / ABC-type xenobiotic transporter / NFE2L2 regulating MDR associated enzymes / Differentiation of Keratinocytes in Interfollicular Epidermis in Mammalian Skin / cellular detoxification / Heme biosynthesis / ABC-type xenobiotic transporter activity / Heme degradation / efflux transmembrane transporter activity / xenobiotic transmembrane transporter activity / ATPase-coupled transmembrane transporter activity / transport across blood-brain barrier / Iron uptake and transport / brush border membrane / mitochondrial membrane / transmembrane transport / apical plasma membrane / membrane raft / protein homodimerization activity / ATP hydrolysis 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
Broad substrate specificity ATP-binding cassette transporter ABCG2
Similarity search - Component
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.12 Å
AuthorsKowal J / Locher K
Funding support Switzerland, 1 items
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, 2021-
Header (metadata) releaseApr 21, 2021-
Map releaseApr 21, 2021-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0121
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0121
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7neq
  • Surface level: 0.0121
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12290.png

Downloads & links

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Map

FileDownload / File: emd_12290.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHuman ABCG2 with tariquidar substrate map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.66 Å/pix.
x 384 pix.
= 253.44 Å		0.66 Å/pix.
x 384 pix.
= 253.44 Å		0.66 Å/pix.
x 384 pix.
= 253.44 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.66 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0121 / Movie #1: 0.0121
Minimum - Maximum-0.050930243 - 0.088107996
Average (Standard dev.)0.00010846318 (±0.0017406092)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 253.44 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.660.660.66
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z253.440253.440253.440
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.0510.0880.000

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Supplemental data

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Additional map: Human ABCG2 with tariquidar substrate map, processed in CryoSPARC

Fileemd_12290_additional_1.map
AnnotationHuman ABCG2 with tariquidar substrate map, processed in CryoSPARC
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Multidrug transporter ABCG2 with bound substrate tariquidar and 5...

EntireName: Multidrug transporter ABCG2 with bound substrate tariquidar and 5D3-Fab variable domain
Components
  • Complex: Multidrug transporter ABCG2 with bound substrate tariquidar and 5D3-Fab variable domain
    • Complex: ATP-binding cassette sub-family G member 2
      • Protein or peptide: ATP-binding cassette sub-family G member 2
    • Complex: 5D3(Fab) light- and heavy-chain variable domains
      • Protein or peptide: 5D3(Fab) light chain variable domain
      • Protein or peptide: 5D3(Fab) heavy chain variable domain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: [(2~{S})-3-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-2-decanoyloxy-propyl] octadecanoate
  • Ligand: CHOLESTEROL
  • Ligand: tariquidar
  • Ligand: water

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Supramolecule #1: Multidrug transporter ABCG2 with bound substrate tariquidar and 5...

SupramoleculeName: Multidrug transporter ABCG2 with bound substrate tariquidar and 5D3-Fab variable domain
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Molecular weightTheoretical: 250 KDa

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Supramolecule #2: ATP-binding cassette sub-family G member 2

SupramoleculeName: ATP-binding cassette sub-family G member 2 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: 5D3(Fab) light- and heavy-chain variable domains

SupramoleculeName: 5D3(Fab) light- and heavy-chain variable domains / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: ATP-binding cassette sub-family G member 2

MacromoleculeName: ATP-binding cassette sub-family G member 2 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: ABC-type xenobiotic transporter
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 72.385852 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSSSNVEVFI PVSQGNTNGF PATASNDLKA FTEGAVLSFH NICYRVKLKS GFLPCRKPVE KEILSNINGI MKPGLNAILG PTGGGKSSL LDVLAARKDP SGLSGDVLIN GAPRPANFKC NSGYVVQDDV VMGTLTVREN LQFSAALRLA TTMTNHEKNE R INRVIQEL ...String:
MSSSNVEVFI PVSQGNTNGF PATASNDLKA FTEGAVLSFH NICYRVKLKS GFLPCRKPVE KEILSNINGI MKPGLNAILG PTGGGKSSL LDVLAARKDP SGLSGDVLIN GAPRPANFKC NSGYVVQDDV VMGTLTVREN LQFSAALRLA TTMTNHEKNE R INRVIQEL GLDKVADSKV GTQFIRGVSG GERKRTSIGM ELITDPSILF LDEPTTGLDS STANAVLLLL KRMSKQGRTI IF SIHQPRY SIFKLFDSLT LLASGRLMFH GPAQEALGYF ESAGYHCEAY NNPADFFLDI INGDSTAVAL NREEDFKATE IIE PSKQDK PLIEKLAEIY VNSSFYKETK AELHQLSGGE KKKKITVFKE ISYTTSFCHQ LRWVSKRSFK NLLGNPQASI AQII VTVVL GLVIGAIYFG LKNDSTGIQN RAGVLFFLTT NQCFSSVSAV ELFVVEKKLF IHEYISGYYR VSSYFLGKLL SDLLP MRML PSIIFTCIVY FMLGLKPKAD AFFVMMFTLM MVAYSASSMA LAIAAGQSVV SVATLLMTIC FVFMMIFSGL LVNLTT IAS WLSWLQYFSI PRYGFTALQH NEFLGQNFCP GLNATGNNPC NYATCTGEEY LVKQGIDLSP WGLWKNHVAL ACMIVIF LT IAYLKLLFLK KYS

UniProtKB: Broad substrate specificity ATP-binding cassette transporter ABCG2

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

MacromoleculeName: 5D3(Fab) light chain variable domain / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.594016 KDa
Recombinant expressionOrganism: Mus musculus (house mouse)
SequenceString: DIVLTQSPSS FSVSLGDRVT ISCKASGYIL NRLAWYQQKP GNAPRLLISG ATSLETGFPS RFSGTGSGKD YTLSISSLQT EDVGTYYCQ QYWSTPWTFG GGTKLEIRRA DAAPTVSIFP PSSEQLTSGG ASVVCFLNNF YPKDINVKWK IDGSERQNGV L NSWTDQDS ...String:
DIVLTQSPSS FSVSLGDRVT ISCKASGYIL NRLAWYQQKP GNAPRLLISG ATSLETGFPS RFSGTGSGKD YTLSISSLQT EDVGTYYCQ QYWSTPWTFG GGTKLEIRRA DAAPTVSIFP PSSEQLTSGG ASVVCFLNNF YPKDINVKWK IDGSERQNGV L NSWTDQDS KDSTYSMSST LTLTKDEYER HNSYTCEATH KTSTSPIVKS FNRNEC

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Macromolecule #3: 5D3(Fab) heavy chain variable domain

MacromoleculeName: 5D3(Fab) heavy chain variable domain / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.843633 KDa
Recombinant expressionOrganism: Mus musculus (house mouse)
SequenceString: QVQLQESGPG LVKPSQSLSL TCTVTGFSIT SDYAWNWIRQ FPGKKLEWMG YINFDGGTTY NPSLRGRISI TRDTSKNQFF LQLRSVTPE DTATYYCATF YGAKGTLDYW GQGTSVTVSS AKTTPPSVYP LAPVCGDTSG SSVTLGCLVK GYFPEPVTLT W NSGSLSSG ...String:
QVQLQESGPG LVKPSQSLSL TCTVTGFSIT SDYAWNWIRQ FPGKKLEWMG YINFDGGTTY NPSLRGRISI TRDTSKNQFF LQLRSVTPE DTATYYCATF YGAKGTLDYW GQGTSVTVSS AKTTPPSVYP LAPVCGDTSG SSVTLGCLVK GYFPEPVTLT W NSGSLSSG VHTFPAVLQS DLYTLSSSVT VTSSTWPSQS ITCNVAHPAS STKVDKKIEP RGP

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 2 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #5: [(2~{S})-3-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-2-decanoyloxy-...

MacromoleculeName: [(2~{S})-3-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-2-decanoyloxy-propyl] octadecanoate
type: ligand / ID: 5 / Number of copies: 1 / Formula: U9N
Molecular weightTheoretical: 635.853 Da
Chemical component information

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

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Macromolecule #6: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 6 / Number of copies: 3 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL

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Macromolecule #7: tariquidar

MacromoleculeName: tariquidar / type: ligand / ID: 7 / Number of copies: 1 / Formula: R1H
Molecular weightTheoretical: 646.732 Da
Chemical component information

ChemComp-R1H:
tariquidar / inhibitor*YM

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Macromolecule #8: water

MacromoleculeName: water / type: ligand / ID: 8 / Number of copies: 16 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 80.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.12 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 166727
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationSoftware - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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