Structure of a zosuquidar and UIC2-bound human-mouse chimeric ABCB1

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 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.

Keywords: ABC transporter; cryo-EM; mechanism; small-molecule inhibitors; structure.

Fig. 1.

Structural and functional characterization of UIC2-Fab and zosuquidar-bound ABCB1. (A) Topology diagram of ABCB1. TM helices are numbered, and their relative lengths, as well as the locations of kinks or bends, are schematically illustrated. The locations of residues interacting with bound zosuquidar are schematically shown as filled green circles. External loops EL1, EL3, and EL4 interact with bound UIC2-Fab, as indicated by red lines. C, carboxy terminus; c.h., coupling helices; e.h., elbow helices; N, amino terminus. (B) Inhibition of ABCB1_HM-X–mediated protection from paclitaxel by zosuquidar (n = 3, error bars represent SD). (C) Inhibition of ABCB1_HM-X–mediated protection from paclitaxel by UIC2 (n = 3, error bars represent SD). (D) Ribbon representation of the UIC2-bound ABCB1_HM-X structure, with the two halves of ABCB1 colored yellow and orange, respectively, and the heavy (HC) and light (LC) chains of UIC2-Fab colored blue and red, respectively. The approximate location of the membrane is depicted in gray. Two bound zosuquidar molecules are shown in light blue and pink sphere representation. The engineered disulfide between the two NBDs is shown as black sticks.

Fig. 2.

Details of UIC2-binding interface and zosuquidar-binding pocket. (A) Close-up view of the ABCB1–UIC2 interface with the UIC2-Fab–binding region shown as an electrostatic surface potential map and ABCB1 shown in ribbon representation. TM helices and ELs of ABCB1 are labeled. ABCB1 residues interacting with UIC2-Fab or with a structural role in stabilizing the outward-closed conformation of ABCB1 are shown as sticks and labeled. (B) Similar view as in A, but with ABCB1 shown as semitransparent electrostatic surface potential map. UIC2-Fab is shown in ribbon representation and colored blue (heavy chain) or red (light chain). Select UIC2 residues within 5 Å of ABCB1 are shown as sticks. (C) Sequence alignments of EL1, EL3, and EL4 regions of ABCB1_H and mouse ABCB1, with secondary structure motifs shown above the sequences. Black dots represent residues of ABCB1_H within 4 Å of UIC2 as seen in our ABCB1_HM-X–UIC2 structure. (D) Ribbon diagram of ABCB1 viewed parallel to the membrane plane, with several TM helices removed for clarity. Two bound zosuquidar molecules are shown as pink and blue sticks, and the corresponding EM density is shown as pink and blue mesh. Selected ABCB1 residues within 4 Å distance of bound zosuquidar are shown as yellow sticks and labeled. (E) Similar to D but viewed from the external side of the membrane.

Fig. 3.

Conformational changes in ABCB1. (A) Comparison of apo-inward ABCB1_HM–UIC2 (blue) and disulfide-trapped, apo ABCB1_HM-X–UIC2 (yellow) using UIC2-Fab as an anchor point for the superposition. The gray box represents the approximate location of the plasma membrane. (B) Close-up view of the NBDs of the two structures shown in A, but using NBD1 as the superposition anchor. The coupling helices of ABCB1_HM and ABCB1_HM-X are colored red and black, respectively. The Cα atoms of V264 and V908 of the coupling helices are represented as spheres, with the distance between them shown as black and red arrows. (C) Superposition of the TM helix pairs TM3–TM4 and TM9–TM10 of the two ABCB1 structures colored as in A. Red and green spheres depict Cα atoms of selected residues, with numbers indicated. A sequence alignment of TM4 and TM10 of human ABCB1 is shown below the structures, and the designated residues are indicated with arrows.

Fig. 4.

Structural changes in the translocation pathway. (A) Surface representations of inward-open ABCB1_HM and disulfide-trapped ABCB1_HM-X structures (without bound UIC2-Fab) colored blue and yellow, respectively. Internal cavity volumes are depicted as dark blue and gold-colored surfaces. The dashed oval indicates the location of the occluded TM cavity that binds zosuquidar in the zosuquidar-bound structure. The gray box represents the membrane. (B) Translocation pathways of ABCB1_HM and ABCB1_HM-X colored as in A, but viewed from the cytoplasmic side of the membrane. TM helices are shown as ribbons and numbered. Arrows indicate the constriction point or cavity gate formed by kinks in TM4 and TM10, closing off the cavity to the cytoplasm in the occluded ABCB1_HM-X structure. (C) Side views of ribbon representation of TM4 and TM10 and the corresponding EM density of ABCB1_HM-X structures. (Left and Center) Two populations of disulfide-trapped apo ABCB1_HM-X, suggesting equilibrium between kinked (closed) and straight (open) conformations. (Right) Zosuquidar-bound structure, where only the kinked conformation exists. Density for the bound zosuquidar molecules is shown as red mesh.