Multidrug resistance conferred by ABC transporters is achieved by efflux of a wide range of drugs from cells, which reduces the intracellular drug concentration and consequently causes drug insensitivity. Human ABCB1(P-gp) is first identified for its role in conferring multidrug resistance in cancer cells and later found to be highly expressed in physiological barriers such as the blood-brain barrier (BBB), where it protects critical organs from xenobiotics accumulation. In aquatic animals such as zebrafish, Abcb4, a functional homolog to hP-gp, plays a vital role in surviving environmental toxicants. It is highly expressed in embryo, adult BBB and digestive system to perform P-gp-like efflux activity. Sharing 64% sequence identity and an overlapping substrate specificity profile with P-gp, zebrafish Abcb4 offers a potential vertebrate model for studying BBB drug penetration and P-gp-mediated multidrug resistance in vivo. To understand the structure and function of zebrafish Abcb4, recombinant wildtype Abcb4 is tested for its ATPase activity, which showed similar basal and drug-stimulated ATPase activity profile as P-gp. Using cryo-EM, we captured five inward-facing Abcb4 conformations with varying separations between its two lobes, illustrating its open-and-close motion, which forms the basis of its basal ATPase activity. The range of separation exceeds that seen in published P-gp structures that appear to be conformationally restricted by either crosslinking modifications, hydrolysis-defective mutations, conformation-selective antibodies, or nanodisc-embedment. The global open-and-close motion drives individual transmembrane helix movements that include twists, tilting, and deformation, which effectively changes the surface topology of the substrate binding pocket encompassed by the transmembrane helices. The highly fluid substrate-binding pocket likely underlies the polyspecificity of substrate recognition, predicts unconventional protein-ligand interactions that are supported by structures of Abcb4 bound to the P-gp inhibitors tariquidar and elacridar, and the substrate vincristine.
Zhan et al. (Sun,) studied this question.