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- PDB-7oj8: ABCG2 E1S turnover-2 state -

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Basic information

Entry
Database: PDB / ID: 7oj8
TitleABCG2 E1S turnover-2 state
ComponentsBroad substrate specificity ATP-binding cassette transporter ABCG2
KeywordsTRANSPORT PROTEIN / ABC transporter plasma membrane ATP multidrug resistance
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 / Ciprofloxacin ADME / transepithelial transport / 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
ADENOSINE-5'-TRIPHOSPHATE / CHOLESTEROL / estrone 3-sulfate / Broad substrate specificity ATP-binding cassette transporter ABCG2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsNi, D. / Yu, Q. / Kowal, J. / Manolaridis, I. / Jackson, S.M. / Stahlberg, H. / Locher, K.P.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science Foundationnot applicable Switzerland
CitationJournal: Nat Commun / Year: 2021
Title: Structures of ABCG2 under turnover conditions reveal a key step in the drug transport mechanism.
Authors: Qin Yu / Dongchun Ni / Julia Kowal / Ioannis Manolaridis / Scott M Jackson / Henning Stahlberg / Kaspar P Locher /
Abstract: ABCG2 is a multidrug transporter that affects drug pharmacokinetics and contributes to multidrug resistance of cancer cells. In previously reported structures, the reaction cycle was halted by the ...ABCG2 is a multidrug transporter that affects drug pharmacokinetics and contributes to multidrug resistance of cancer cells. In previously reported structures, the reaction cycle was halted by the absence of substrates or ATP, mutation of catalytic residues, or the presence of small-molecule inhibitors or inhibitory antibodies. Here we present cryo-EM structures of ABCG2 under turnover conditions containing either the endogenous substrate estrone-3-sulfate or the exogenous substrate topotecan. We find two distinct conformational states in which both the transport substrates and ATP are bound. Whereas the state turnover-1 features more widely separated NBDs and an accessible substrate cavity between the TMDs, turnover-2 features semi-closed NBDs and an almost fully occluded substrate cavity. Substrate size appears to control which turnover state is mainly populated. The conformational changes between turnover-1 and turnover-2 states reveal how ATP binding is linked to the closing of the cytoplasmic side of the TMDs. The transition from turnover-1 to turnover-2 is the likely bottleneck or rate-limiting step of the reaction cycle, where the discrimination of substrates and inhibitors occurs.
History
DepositionMay 14, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 21, 2021Provider: repository / Type: Initial release
Revision 1.1Jul 28, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

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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)149,9659
Polymers147,0542
Non-polymers2,9117
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area9800 Å2
ΔGint-41 kcal/mol
Surface area47800 Å2
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "A"
d_2ens_1(chain "B" and (resid 34 through 654 or resid 1501))

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1GLYTYRA1 - 575
d_12ens_1ATPATPB
d_21ens_1GLYTYRC1 - 575
d_22ens_1ATPATPD

NCS oper: (Code: givenMatrix: (-0.979803122974, -0.0471204427481, -0.194333486783), (-0.0344090138291, -0.917613671633, 0.395981274052), (-0.196981877282, 0.394670512589, 0.897459373184)Vector: 82. ...NCS oper: (Code: given
Matrix: (-0.979803122974, -0.0471204427481, -0.194333486783), (-0.0344090138291, -0.917613671633, 0.395981274052), (-0.196981877282, 0.394670512589, 0.897459373184)
Vector: 82.5923889185, 72.9048808727, -6.75493331578)

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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 / Cell line (production host): HEK293-EBNA / Production host: Homo sapiens (human)
References: UniProt: Q9UNQ0, ABC-type xenobiotic transporter
#2: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


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


Mass: 386.654 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C27H46O
#4: Chemical ChemComp-FY5 / estrone 3-sulfate / Estrone sulfate


Mass: 350.429 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C18H22O5S / Feature type: SUBJECT OF INVESTIGATION / Comment: medication, hormone*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 E1S under turnover condition / Type: COMPLEX
Details: ABCG2 was incubated with 5mM ATP, 5mM MgCl2, 0.5mM ADP, 200 uM E1S at room temperature for 10 min
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.144 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293EBNA
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
10.15 MNaClSodium chloride1
20.04 MHepes1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: The sample was mono-disperse.
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 / Details: Grids were blotted for 2.5s with blot force 1

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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: DARK FIELD / Nominal magnification: 165000 X / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsEnergyfilter name: GIF Quantum LS / Details: no phase plate / Energyfilter slit width: 20 eV

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.18.2_3874refinement
PHENIX1.18.2_3874refinement
EM software
IDNameVersionCategory
1cryoSPARC2.1particle selection
2SerialEM2image acquisition
4cryoSPARC2.1CTF correction
7UCSF Chimeramodel fitting
10cryoSPARC2.1final Euler assignment
11cryoSPARC2.1classification
13PHENIXmodel refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1437275
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 221160 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 6HCO

6hco

RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 96.14 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00829355
ELECTRON MICROSCOPYf_angle_d0.875112703
ELECTRON MICROSCOPYf_chiral_restr0.05211478
ELECTRON MICROSCOPYf_plane_restr0.00431544
ELECTRON MICROSCOPYf_dihedral_angle_d15.04343340
Refine LS restraints NCSType: NCS constraints / Rms dev position: 0.100870226101 Å

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