Journal: Proc. Natl. Acad. Sci. U.S.A. / Year: 2018 Title: Structure of a zosuquidar and UIC2-bound human-mouse chimeric ABCB1. Authors: Amer Alam / Raphael Küng / Julia Kowal / Robert A McLeod / Nina Tremp / Eugenia V Broude / Igor B Roninson / Henning Stahlberg / Kaspar P Locher Abstract: The multidrug transporter ABCB1 (P-glycoprotein) is an ATP-binding cassette transporter that has a key role in protecting tissues from toxic insult and contributes to multidrug extrusion from cancer ...The multidrug transporter ABCB1 (P-glycoprotein) is an ATP-binding cassette transporter that has a key role in protecting tissues from toxic insult and contributes to multidrug extrusion from cancer cells. Here, we report the near-atomic resolution cryo-EM structure of nucleotide-free ABCB1 trapped by an engineered disulfide cross-link between the nucleotide-binding domains (NBDs) and bound to the antigen-binding fragment of the human-specific inhibitory antibody UIC2 and to the third-generation ABCB1 inhibitor zosuquidar. Our structure reveals the transporter in an occluded conformation with a central, enclosed, inhibitor-binding pocket lined by residues from all transmembrane (TM) helices of ABCB1. The pocket spans almost the entire width of the lipid membrane and is occupied exclusively by two closely interacting zosuquidar molecules. The external, conformational epitope facilitating UIC2 binding is also visualized, providing a basis for its inhibition of substrate efflux. Additional cryo-EM structures suggest concerted movement of TM helices from both halves of the transporters associated with closing the NBD gap, as well as zosuquidar binding. Our results define distinct recognition interfaces of ABCB1 inhibitory agents, which may be exploited for therapeutic purposes.
Date
Deposition: Feb 2, 2018 / Header (metadata) release: Feb 14, 2018 / Map release: Feb 21, 2018 / Last update: Mar 7, 2018
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Structure visualization
Movie
Surface view with section colored by density value
Entire Apo Human-mouse chimeric ABCB1 (ABCB1HM) complex with UIC2 fab
Entire
Name: Apo Human-mouse chimeric ABCB1 (ABCB1HM) complex with UIC2 fab Number of components: 3
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Component #1: protein, Apo Human-mouse chimeric ABCB1 (ABCB1HM) complex with UI...
Protein
Name: Apo Human-mouse chimeric ABCB1 (ABCB1HM) complex with UIC2 fab Recombinant expression: No
Mass
Theoretical: 195 kDa
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Component #2: protein, Apo Human-mouse chimeric ABCB1 (ABCB1HM)
Protein
Name: Apo Human-mouse chimeric ABCB1 (ABCB1HM) / Recombinant expression: No
Mass
Theoretical: 142 kDa
Source
Species: Homo sapiens (human)
Source (engineered)
Expression System: Homo sapiens (human)
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Component #3: protein, UIC2 fab
Protein
Name: UIC2 fab / Recombinant expression: No
Mass
Theoretical: 48.7 kDa
Source
Species: Mus musculus (house mouse)
Source (engineered)
Expression System: Mus musculus (house mouse)
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Experimental details
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Sample preparation
Specimen
Specimen state: particle / Method: cryo EM
Sample solution
pH: 7.5
Vitrification
Cryogen name: ETHANE
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Electron microscopy imaging
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
Imaging
Microscope: FEI TITAN KRIOS
Electron gun
Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 0.9 e/Å2 / Illumination mode: FLOOD BEAM
Lens
Imaging mode: BRIGHT FIELD
Specimen Holder
Model: OTHER
Camera
Detector: GATAN K2 SUMMIT (4k x 4k)
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Image processing
Processing
Method: single particle reconstruction / Number of projections: 153652
3D reconstruction
Resolution: 4.47 Å / Resolution method: FSC 0.143 CUT-OFF
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