Functional Implications of the Conformational Landscape of a Multidrug Transporter Revealed by Structures of Zebrafish Abcb4

Abstract The hallmark of multidrug resistance conferred by human ABC transporter ABCB1 (hP-gp) is the recognition and efflux of diverse range of drugs, though the precise mechanism of polyspecificity remains unresolved. In aquatic animals such as zebrafish, Abcb4, a functional homolog to hP-gp, plays a vital role in surviving environmental toxicants. Here, we show that Abcb4 exhibits comparable basal and drug-stimulated ATPase activity to hP-gp. Using cryo-EM, we captured five inward-facing Abcb4 conformations with varying separations between its two lobes, illustrating its open-and- close motion. The range of separation exceeds that seen in published P-gp structures that appear to be conformationally restricted. This global open-and-close motion is coupled with individual helix movement, resulting in a highly fluid substrate-binding pocket. These dynamic changes, likely underlying the polyspecificity of substrate recognition, predict unconventional protein-ligand interactions that are supported by structures of Abcb4 bound to the P-gp inhibitors tariquidar and elacridar, and the substrate vincristine. Competing Interest Statement The authors have declared no competing interest. List of abbreviations - ABC - ATP-Binding Cassette - DCOM - Distance between center of mass of the two NBDs - IF - inward facing - OF - outward facing - P-gp - P-glycoprotein or Permeability glycoprotein - hP-gp - human P-gp - mP-gp - murine P-gp - TM - transmembrane - TMH - TM helix