Structural insight into substrate and inhibitor discrimination by human P-glycoprotein

Abstract

ABCB1, also known as P-glycoprotein, actively extrudes xenobiotic compounds across the plasma membrane of diverse cells, which contributes to cellular drug resistance and interferes with therapeutic drug delivery. We determined the 3.5-angstrom cryo-electron microscopy structure of substrate-bound human ABCB1 reconstituted in lipidic nanodiscs, revealing a single molecule of the chemotherapeutic compound paclitaxel (Taxol) bound in a central, occluded pocket. A second structure of inhibited, human-mouse chimeric ABCB1 revealed two molecules of zosuquidar occupying the same drug-binding pocket. Minor structural differences between substrate- and inhibitor-bound ABCB1 sites are amplified toward the nucleotide-binding domains (NBDs), revealing how the plasticity of the drug-binding site controls the dynamics of the adenosine triphosphate-hydrolyzing NBDs. Ordered cholesterol and phospholipid molecules suggest how the membrane modulates the conformational changes associated with drug binding and transport.

Fig. 1

In vitro function and structure of nanodisc-reconstituted ABCB1. A Taxol-and zosuquidar-modulated ATPase activity (n=3, error bars indicate SD). B Ribbon diagram of human ABCB1 bound to taxol (green spheres). The N-and C-terminal halves of ABCB1 are colored yellow and orange, respectively with the UIC2 Fab shown in in blue. C Close up of binding site showing side chains of residues within 5Å of bound taxol (green sticks), viewed parallel to the membrane plane. EM density is shown as a blue mesh, contoured at 6 σ. D Ribbon representation of TM4 (yellow) and TM10 (orange) adopting kinked conformation, with taxol located in the center of the occluded cavity. EM density after nanodisc subtraction is contoured at 8 σ. E interactions between taxol and ABCB1 side chains. Non-bonded interactions are represented by spoked arcs and hydrogen bonds are indicated by dashed black lines.

Figure 2.

Comparison of zosuquidar-and taxol-bound ABCB1. A Cartoon of zosuquidar-bound ABCB1_HM-EQ structure with-zosuquidar molecules shown as yellow and magenta spheres. The N-and C-terminal halves of ABCB1 are colored pink and blue. B-D Superposition of taxol-bound human ABCB1 (green ribbon) and zosuquidar-bound ABCB1_HM-EQ (magenta ribbon) with intracellular helices interacting with NBDs shown as cylinders. Zosuquidar molecules are shown in sphere representation.

Figure 3

Phospholipids and cholesterol bound to ABCB1. Center panel: Surface representation of taxol-bound human ABCB1 showing bound lipid (PE, magenta spheres) and cholesterol molecules (purple spheres). Zoom in panels show details of binding sites of phospholipid (left panel, magenta sticks) and cholesterol (right panel, purple sticks) at the level of the inner leaflet, near the kinking TM helices TM4 and TM10. EM density (blue mesh) is contoured at 6σ.

Figure 4

Proposed mechanism of P-glycoprotein / ABCB1 substrate transport and small-molecule inhibition. A Side view of select TM helices of ABCB1 after superposition of the drug-bound, occluded conformation (green and magenta ribbons for taxol-and zosuquidar-bound structures) with the previously reported, ATP-bound post-translocation state (dark grey ribbon). Taxol and zosuquidar molecules are shown as green and red spheres, respectively. B Same as A but viewed from the cytoplasm. C Schematic of proposed ABCB1 transport cycle in the presence of substrate (taxol, green star) and inhibitor (zosuquidar, red L-shape). ATP and ADP are indicated by T and D, respectively. Dashed lines in models represent ATP binding elements required for NBD dimerization and ATP hydrolysis. Major conformational states are represented by circled numbers. State 1: Apo state (pdb IDs 4M1M & 4QNH, among others); States 2 and 2’: Drug-and inhibitor-bound, this study. State 3: Proposed outward-facing conformation based on Sav1866 structure(30). State 4: Collapsed post-translocation state (pdb ID 6C0V).