Journal: 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
Deposition
May 14, 2021
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Header (metadata) release
Jul 21, 2021
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Map release
Jul 21, 2021
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Update
Jul 28, 2021
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Current status
Jul 28, 2021
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Entire : ABCG2 in complex with E1S under turnover condition
Entire
Name: ABCG2 in complex with E1S under turnover condition
Components
Complex: ABCG2 in complex with E1S under turnover condition
Protein or peptide: Broad substrate specificity ATP-binding cassette transporter ABCG2
Ligand: ADENOSINE-5'-TRIPHOSPHATE
Ligand: CHOLESTEROL
Ligand: estrone 3-sulfate
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Supramolecule #1: ABCG2 in complex with E1S under turnover condition
Supramolecule
Name: ABCG2 in complex with E1S under turnover condition / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 Details: ABCG2 was incubated with 5mM ATP, 5mM MgCl2, 0.5mM ADP, 200 uM E1S at room temperature for 10 min
Source (natural)
Organism: Homo sapiens (human)
Recombinant expression
Organism: Homo sapiens (human) / Recombinant cell: HEK293EBNA
Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
Vitrification
Cryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Grids were blotted for 2.5s with blot force 1.
Details
The sample was mono-disperse.
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Electron microscopy
Microscope
FEI TITAN KRIOS
Specialist optics
Energy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV / Details: no phase plate
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 50.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron optics
C2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: DARK FIELD / Cs: 2.7 mm / Nominal magnification: 165000
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