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- PDB-8bht: ABCG2 turnover-1 state with tariquidar bound -

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Basic information

Entry
Database: PDB / ID: 8bht
TitleABCG2 turnover-1 state with tariquidar bound
ComponentsBroad substrate specificity ATP-binding cassette transporter ABCG2
KeywordsTRANSPORT PROTEIN / Multidrug resistance / ABC transporter
Function / homology
Function and homology information


biotin transmembrane transporter activity / biotin transport / riboflavin transport / riboflavin transmembrane transporter activity / renal urate salt excretion / sphingolipid transporter activity / urate metabolic process / urate transmembrane transporter activity / Abacavir transmembrane transport / organic anion transport ...biotin transmembrane transporter activity / biotin transport / riboflavin transport / riboflavin transmembrane transporter activity / renal urate salt excretion / sphingolipid transporter activity / urate metabolic process / urate transmembrane transporter activity / Abacavir transmembrane transport / organic anion transport / external side of apical plasma membrane / sphingolipid biosynthetic process / organic anion transmembrane transporter activity / Sphingolipid de novo biosynthesis / xenobiotic transport across blood-brain barrier / transepithelial transport / export across plasma membrane / ABC-type xenobiotic transporter / Paracetamol ADME / Ciprofloxacin ADME / ABC-type xenobiotic transporter activity / NFE2L2 regulating MDR associated enzymes / cellular detoxification / Heme biosynthesis / Heme degradation / efflux transmembrane transporter activity / xenobiotic 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 / 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
ADENOSINE-5'-TRIPHOSPHATE / CHOLESTEROL / tariquidar / Broad substrate specificity ATP-binding cassette transporter ABCG2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsYu, Q. / Kowal, J. / Tajkhorshid, E. / Locher, K.P.
Funding support Switzerland, United States, 3items
OrganizationGrant numberCountry
Swiss National Science FoundationNCCR TransCure Switzerland
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41- GM104601 United States
National Institutes of Health/Office of the DirectorR01-GM123455 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Differential dynamics and direct interaction of bound ligands with lipids in multidrug transporter ABCG2.
Authors: Ali Rasouli / Qin Yu / Sepehr Dehghani-Ghahnaviyeh / Po-Chao Wen / Julia Kowal / Kaspar P Locher / Emad Tajkhorshid /
Abstract: ABCG2 is an ATP-binding cassette (ABC) transporter that extrudes a wide range of xenobiotics and drugs from the cell and contributes to multidrug resistance in cancer cells. Following our recent ...ABCG2 is an ATP-binding cassette (ABC) transporter that extrudes a wide range of xenobiotics and drugs from the cell and contributes to multidrug resistance in cancer cells. Following our recent structural characterization of topotecan-bound ABCG2, here, we present cryo-EM structures of ABCG2 under turnover conditions in complex with a special modulator and slow substrate, tariquidar, in nanodiscs. The structures reveal that similar to topotecan, tariquidar induces two distinct ABCG2 conformations under turnover conditions (turnover-1 and turnover-2). μs-scale molecular dynamics simulations of drug-bound and apo ABCG2 in native-like lipid bilayers, in both topotecan- and tariquidar-bound states, characterize the ligand size as a major determinant of its binding stability. The simulations highlight direct lipid-drug interactions for the smaller topotecan, which exhibits a highly dynamic binding mode. In contrast, the larger tariquidar occupies most of the available volume in the binding pocket, thus leaving little space for lipids to enter the cavity and interact with it. Similarly, when simulating ABCG2 in the apo inward-open state, we also observe spontaneous penetration of phospholipids into the binding cavity. The captured phospholipid diffusion pathway into ABCG2 offers a putative general path to recruit any hydrophobic/amphiphilic substrates directly from the membrane. Our simulations also reveal that ABCG2 rejects cholesterol as a substrate, which is omnipresent in plasma membranes that contain ABCG2. At the same time, cholesterol is found to prohibit the penetration of phospholipids into ABCG2. These molecular findings have direct functional ramifications on ABCG2's function as a transporter.
History
DepositionOct 31, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 11, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Broad substrate specificity ATP-binding cassette transporter ABCG2
B: Broad substrate specificity ATP-binding cassette transporter ABCG2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)149,4887
Polymers147,0542
Non-polymers2,4345
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area7780 Å2
ΔGint-43 kcal/mol
Surface area46860 Å2
MethodPISA
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1(chain "A" and resid 34 through 654)
d_2ens_1(chain "B" and resid 34 through 654)

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1GLYTYRA1 - 569
d_21ens_1GLYTYRE1 - 569

NCS oper: (Code: givenMatrix: (-0.542997149003, 0.116248923929, 0.831649135069), (0.178247531954, -0.951846631469, 0.249430967429), (0.820598509338, 0.283679709965, 0.496128923393)Vector: 9. ...NCS oper: (Code: given
Matrix: (-0.542997149003, 0.116248923929, 0.831649135069), (0.178247531954, -0.951846631469, 0.249430967429), (0.820598509338, 0.283679709965, 0.496128923393)
Vector: 9.73683952454, 57.6684818988, -16.1768360186)

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Components

#1: Protein Broad substrate specificity ATP-binding cassette transporter ABCG2 / ATP-binding cassette sub-family G member 2 / Breast cancer resistance protein / CDw338 / ...ATP-binding cassette sub-family G member 2 / Breast cancer resistance protein / CDw338 / Mitoxantrone resistance-associated protein / Placenta-specific ATP-binding cassette transporter / Urate exporter


Mass: 73526.938 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) / Strain (production host): HEK293-EBNA
References: UniProt: Q9UNQ0, ABC-type xenobiotic transporter
#2: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#3: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H46O / Feature type: SUBJECT OF INVESTIGATION
#4: 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


Mass: 646.732 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C38H38N4O6 / Feature type: SUBJECT OF INVESTIGATION / Comment: inhibitor*YM
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

ComponentName: ABCG2 in complex with Tariquidar under turnover condition
Type: COMPLEX
Details: ABCG2 was incubated with 5mM ATP, 5mM MgCl2, 0.5mM ADP, 20 uM Tariquidar at room temperature for 10 min
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.144 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Strain: HEK293EBNA
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
140 mMHEPESC8H18N2O4S1
2150 mMNaCl1
31
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was mono-disperse
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 / Humidity: 100 % / Chamber temperature: 277.15 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 / Nominal magnification: 130000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.49 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18.2_3874refinement
PHENIX1.18.2_3874refinement
EM software
IDNameVersionCategory
1cryoSPARC2.15particle selection
2EPU2image acquisition
4GctfCTF correction
7Coot0.9.2model fitting
9cryoSPARC2.15initial Euler assignment
10cryoSPARC2.15final Euler assignment
11cryoSPARC2.15classification
12cryoSPARC2.153D reconstruction
13PHENIX1.18.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2939835
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 211906 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL / Target criteria: cross-correlation coefficient
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 48.16 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00729217
ELECTRON MICROSCOPYf_angle_d0.653112500
ELECTRON MICROSCOPYf_chiral_restr0.03991438
ELECTRON MICROSCOPYf_plane_restr0.0031534
ELECTRON MICROSCOPYf_dihedral_angle_d13.44163299
Refine LS restraints NCSType: NCS constraints / Rms dev position: 0.109850677646 Å

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