Cryo-EM structures reveal distinct mechanisms of inhibition of the human multidrug transporter ABCB1

Abstract

ABCB1 detoxifies cells by exporting diverse xenobiotic compounds, thereby limiting drug disposition and contributing to multidrug resistance in cancer cells. Multiple small-molecule inhibitors and inhibitory antibodies have been developed for therapeutic applications, but the structural basis of their activity is insufficiently understood. We determined cryo-EM structures of nanodisc-reconstituted, human ABCB1 in complex with the Fab fragment of the inhibitory, monoclonal antibody MRK16 and bound to a substrate (the antitumor drug vincristine) or to the potent inhibitors elacridar, tariquidar, or zosuquidar. We found that inhibitors bound in pairs, with one molecule lodged in the central drug-binding pocket and a second extending into a phenylalanine-rich cavity that we termed the "access tunnel." This finding explains how inhibitors can act as substrates at low concentration, but interfere with the early steps of the peristaltic extrusion mechanism at higher concentration. Our structural data will also help the development of more potent and selective ABCB1 inhibitors.

Keywords: ABC transporter; ABCB1; P-glycoprotein; single-particle cryoelectron microscopy; structure.

Fig. 1.

Functional characterization of human ABCB1. (A) Normalized ATPase activity of nanodisc-reconstituted ABCB1 in the absence (black bars) or presence of different drugs, and in the absence (−) or presence (+) of the Fab fragment of antibody MRK16. The concentration of ATP was 2 mM throughout. n = 3, error bars represent SDs. (B) Normalized ATPase activity of ABCB1 as a function of ATP concentration in the absence or presence of inhibitors, and in the absence (−) or presence (+) of the MRK16-Fab. The points were plotted with nonlinear regression of the Michaelis–Menten equation. Relative V_max and apparent K_m are displayed in the table. n = 3, error bars represent SDs.

Fig. 2.

Overview of ABCB1–MRK16-Fab structures. (A) Ribbon diagrams of drug-free and drug-bound states of ABCB1–MRK16-Fab structures. The drug molecules are represented in sphere representation. (B) Close-up view of the binding pocket of vincristine- and inhibitor-bound structures, with adjacent TM helices shown as backbone splines and labeled. The thresholds of the EM density maps were adjusted such that the level of density covering the surrounding TM helices is comparable. Drug molecules are shown as sticks and their corresponding chemical structures are shown underneath.

Fig. 3.

ABCB1–MRK16-Fab interface. (A) Ribbon diagram of vincristine-bound ABCB1–MRK16-Fab complex (ABCB1 colored cyan) superimposed with the taxol-bound ABCB1–UIC2-Fab structure [PDB: 6qex (21), ABCB1 colored olive]. (B) Close-up of the ABCB1–MRK16 interface. ABCB1 is shown as a cyan ribbon except for residues in contact with MRK16, which are shown as sticks. The EM density of the ABCB1 residues in contact with MRK16 is shown as a blue mesh. MRK16-Fab is shown as a transparent electrostatic surface (blue, positive charges; red, negative charges). Transmembrane helices, extracellular loops, and ABCB1 residues are labeled. (C) Close-up of TM1 of ABCB1 at the interface with MRK16 (colored as in A). EM density (blue mesh) is shown for MRK16 residues in contact with ABCB1 residues of TM1. Note that the arginine residue R101 causes an unwinding of TM1 of ABCB1.

Fig. 4.

Access tunnel and vestibule in the occluded conformation of ABCB1. (A) Surface representation of ABCB1 structures presented in this study and taxol-bound ABCB1 (PDB: 6qex) (21). The view is parallel to the membrane and the section shown covers the central drug-binding pocket and the cavities (vestibule and access tunnel). These cavities are indicated with dashed lines. (B) Surface and ribbon representation of elacridar-bound ABCB1–MRK16-Fab structure showing the general architecture of the central cavity in the occluded state. The access tunnel extends toward the cytoplasmic side of the membrane allowing water molecules to enter the cavity (black arrow). (C) Schematic of the drug-binding pocket, vestibule, and access tunnel within ABCB1. The transmembrane helices that surround the access tunnel are shown as cylinders. The dashed area corresponds to the areas shown in A.

Fig. 5.

Rigid modules and mobile TM helices facilitate the ABCB1 transport mechanism. (A and B) Superposition of vincristine-bound ABCB1 in an occluded conformation (cyan, this study) and collapsed conformation of ATP-bound ABCB1-EQ mutant (pink, PDB: 6c0v) (22) identified two rigid modules shown as gray and yellow surface, respectively, and four TM domains with distinct conformations. (A) Superposition of five TM domains and NBD1 that define rigid module 1, with no rearrangement seen between the occluded and collapsed conformations (subpanel I). In subpanels II to IV, rigid module 1 is shown as a gray surface and the distinct conformations of TM10, TM12, and TM9 are shown individually in the two states. Subpanel IV also shows the large conformational changes caused by the dimerization of the NBDs. (B) Superposition of three TM domains and NBD2 that define rigid module 2, with no rearrangement seen between the occluded and collapsed conformations (subpanel I). In subpanel II, rigid module 2 is shown as a yellow surface and the distinct conformations of TM4 are shown. (C) Comparison of occluded and collapsed states, with the rigid modules represented as transparent surfaces colored as in A and B and mobile TM domains shown as ribbons and labeled. The view is as in A. (D) Role of TM9: Following the superposition of structures, rigid module 1 is shown as a gray surface, TM9 is shown as a ribbon, and substrates (vincristine, taxol) or inhibitors (elacridar, tariquidar, zosuquidar) are shown as sticks. This shows that the shift of TM9 is prevented by bound inhibitors but possible in the presence of substrates.