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- PDB-6feq: Structure of inhibitor-bound ABCG2 -

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Basic information

Entry
Database: PDB / ID: 6feq
TitleStructure of inhibitor-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 / Multidrug transporter / ABCG2 / MB136 / inhibitor
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 / external side of apical plasma membrane ...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 / external side of apical plasma membrane / sphingolipid biosynthetic process / organic anion transport / Sphingolipid de novo biosynthesis / organic anion transmembrane transporter activity / xenobiotic transport across blood-brain barrier / transepithelial transport / export across plasma membrane / ABC-type xenobiotic transporter / Paracetamol ADME / Ciprofloxacin ADME / NFE2L2 regulating MDR associated enzymes / ABC-type xenobiotic transporter activity / Differentiation of keratinocytes in interfollicular epidermis in mammalian skin / cellular detoxification / Heme biosynthesis / Heme degradation / efflux transmembrane transporter activity / xenobiotic transmembrane transporter activity / ATPase-coupled transmembrane transporter activity / transport across blood-brain barrier / mitochondrial membrane / brush border membrane / Iron uptake and transport / transmembrane transport / membrane raft / apical plasma membrane / 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 ...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 / Immunoglobulins / Immunoglobulin-like / Sandwich / P-loop containing nucleoside triphosphate hydrolase / Mainly Beta
Similarity search - Domain/homology
Chem-D6T / 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: 3.6 Å
AuthorsJackson, S.M. / Manolaridis, I. / Kowal, J. / Zechner, M. / Altmann, K.H. / Locher, K.P.
Funding support Switzerland, 2items
OrganizationGrant numberCountry
Swiss National Science Foundation, National Centre of Competence in Research (NCCR) TransCure Switzerland
Swiss Federal Institute of Technology Zurich (ETH Zurich)ETH-22-14-1 Switzerland
CitationJournal: Nat Struct Mol Biol / Year: 2018
Title: Structural basis of small-molecule inhibition of human multidrug transporter ABCG2.
Authors: Scott M Jackson / Ioannis Manolaridis / Julia Kowal / Melanie Zechner / Nicholas M I Taylor / Manuel Bause / Stefanie Bauer / Ruben Bartholomaeus / Guenther Bernhardt / Burkhard Koenig / ...Authors: Scott M Jackson / Ioannis Manolaridis / Julia Kowal / Melanie Zechner / Nicholas M I Taylor / Manuel Bause / Stefanie Bauer / Ruben Bartholomaeus / Guenther Bernhardt / Burkhard Koenig / Armin Buschauer / Henning Stahlberg / Karl-Heinz Altmann / Kaspar P Locher /
Abstract: ABCG2 is an ATP-binding cassette (ABC) transporter that protects tissues against xenobiotics, affects the pharmacokinetics of drugs and contributes to multidrug resistance. Although many inhibitors ...ABCG2 is an ATP-binding cassette (ABC) transporter that protects tissues against xenobiotics, affects the pharmacokinetics of drugs and contributes to multidrug resistance. Although many inhibitors and modulators of ABCG2 have been developed, understanding their structure-activity relationship requires high-resolution structural insight. Here, we present cryo-EM structures of human ABCG2 bound to synthetic derivatives of the fumitremorgin C-related inhibitor Ko143 or the multidrug resistance modulator tariquidar. Both compounds are bound to the central, inward-facing cavity of ABCG2, blocking access for substrates and preventing conformational changes required for ATP hydrolysis. The high resolutions allowed for de novo building of the entire transporter and also revealed tightly bound phospholipids and cholesterol interacting with the lipid-exposed surface of the transmembrane domains (TMDs). Extensive chemical modifications of the Ko143 scaffold combined with in vitro functional analyses revealed the details of ABCG2 interactions with this compound family and provide a basis for the design of novel inhibitors and modulators.
History
DepositionJan 3, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 11, 2018Provider: repository / Type: Initial release
Revision 1.1Apr 18, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Oct 30, 2019Group: Data collection / Category: em_software / Item: _em_software.name / _em_software.version
Revision 1.3Dec 11, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
Revision 1.4Jul 29, 2020Group: Data collection / Derived calculations / Structure summary
Category: chem_comp / entity ...chem_comp / entity / pdbx_chem_comp_identifier / pdbx_entity_nonpoly / struct_conn / struct_site / struct_site_gen
Item: _chem_comp.name / _chem_comp.type ..._chem_comp.name / _chem_comp.type / _entity.pdbx_description / _pdbx_entity_nonpoly.name / _struct_conn.pdbx_role
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 1.5Oct 23, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp / chem_comp_atom ...chem_comp / chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI ..._chem_comp.pdbx_synonyms / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update

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Assembly

Deposited unit
A: ATP-binding cassette sub-family G member 2
B: 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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)240,8889
Polymers239,6476
Non-polymers1,2413
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area12880 Å2
ΔGint-67 kcal/mol
Surface area64980 Å2
MethodPISA

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Components

#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 / Cell line (production host): HEK293 EBNA / Production host: Homo sapiens (human) / References: UniProt: Q9UNQ0
#2: Antibody 5D3(Fab) light chain variable domain


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


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
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
#5: Chemical ChemComp-D6T / ~{N}-[5-[1-[4-[2-[6-methoxy-7-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]-3,4-dihydro-1~{H}-isoquinolin-2-yl]ethyl]phenyl]-1,2,3-triazol-4-yl]-2-propanoyl-phenyl]quinoline-2-carboxamide


Mass: 798.925 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C46H50N6O7
Has protein modificationY

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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
1inhibitor-bound ABCG2COMPLEX#1-#30NATURAL
2ABCG2COMPLEX#11NATURAL
3inhibitorCOMPLEX#2-#31NATURAL
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12HOMO SAPIENS (human)9606
23MUS MUSCULUS (house mouse)10090
Buffer solutionpH: 7.5
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277 K

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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 FIELD
Image recordingAverage exposure time: 0.25 sec. / Electron dose: 1.55 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 2468

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM softwareName: cryoSPARC / Version: 1 / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 306913 / Symmetry type: POINT
Atomic model buildingB value: 163
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00912716
ELECTRON MICROSCOPYf_angle_d0.9817242
ELECTRON MICROSCOPYf_dihedral_angle_d6.9147425
ELECTRON MICROSCOPYf_chiral_restr0.0561965
ELECTRON MICROSCOPYf_plane_restr0.0062145

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