Understanding polyspecificity of multidrug ABC transporters: closing in on the gaps in ABCB1

Abstract

Multidrug ABC transporters can transport a wide range of drugs from the cell. Ongoing studies of the prototype mammalian multidrug resistance ATP-binding cassette transporter P-glycoprotein (ABCB1) have revealed many intriguing functional and biochemical features. However, a gap remains in our knowledge regarding the molecular basis of its broad specificity for structurally unrelated ligands. Recently, the first crystal structures of ligand-free and ligand-bound ABCB1 showed ligand binding in a cavity between its two membrane domains, and earlier observations on polyspecificity can now be interpreted in a structural context. Comparison of the new ABCB1 crystal structures with structures of bacterial homologs suggests a critical role for an axial rotation of transmembrane helices for high-affinity binding and low-affinity release of ligands during transmembrane transport.

Figure 1

Model for ligand efflux by ABCB1. ABCB1 contains a central ligand-binding cavity close to the leaflet–leaflet interface of the membrane. During transport, this binding cavity is alternately exposed to the inside and outside surface of the membrane. The inward-facing structure of ABCB1 (A) and putative outward-facing structure (B) have a laterally open cleft in their MDs exposed to the lipid phase of the bilayer. In the inward-facing conformation, this “gap” could grant ligand (green) access to the binding site directly from the inner leaflet of the phospholipid bilayer and cytoplasm. When the transporter assumes its outward-facing conformation the ligand is expelled into the outer leaflet and/or aqueous extracellular medium. The outward-facing ABCB1 model in (B) is based on MsbA from S. thyphimurium and Sav1866 from S. aureus. (C) Cross section through ABCB1 bound to two molecules of QZ59-SSS (black) reveals the central location of the ligand-binding site in relation to the phospholipid bilayer. The residues in ABCB1 corresponding to Ser289 and Ser290 in E. coli MsbA, which affect ligand specificity of this bacterial transporter, are shown as brown spheres to highlight their juxtaposition to the bound ligand. The surface is rendered by hydrophobicity (orange) and hydrophilicity (blue). The model in (B) was generated using Modeller. Figure was generated in Chimera.

Figure 2

ABCB1 can use different combinations of flexible side chains in its ligand-binding cavity to create binding sites for different ligands. Structure of ABCB1 complexed with one molecule of QZ59-RRR (red) or two molecules of QZ59-SSS (black). Superimposition of the ligand-binding sites: residues interacting with QZ59-RRR are rendered in red whereas residues interacting with QZ59-SSS are rendered in black. The magnified stereo view (inset) reveals alternative side chain rotamers in the two different drug bound crystal structures. Up to 60 % of the ABCB1–CPPI interactions in the binding cavity is based on interactions with Phe and Tyr side chains. The use of different sets of Phe and Tyr residues in binding of QZ-RRR and QZ59-SSS, and flexibility of these aromatic side chains contribute to the specificity of the ligand-binding cavity for different CPPIs. The N-terminal half of ABCB1 is rendered in green, the C-terminal half in blue. Figure was generated in Pymol.

Figure 3

Stereo views of inward-facing ligand-binding pockets. Views are shown for (A) ABCB1a (mouse), (B) ABCB1 (human), (C) ABCB4 (human) (D) MsbA (E. coli), (E) Sav1866 (S. aureus), and (F) LmrA (L. lactis). Aromatic residues (Tyr, Phe, Trp) are colored red, hydrophobic residues (Gly, Ala, Leu, Ile, Val, Met) are blue and all other residues are colored white. The bacterial models (Sav1866 and LmrA) were based on a full model of MsbA (E. coli) and the mammalian models (ABCB1 and ABCB4) were based on the crystal structure of ABCB1a. The models were created using Swiss Model.

Figure 4

Conformational switch in ABCB1 from the inward to the outward facing state is accompanied by rotation of the transmembrane helices. Helix rotations are evident from a comparison of positions of atoms that comprise helices and bulky amino acid side chains. During the rotation, side chains important for ligand binding to the inward-facing conformation move away from the binding cavity. As favourable protein–ligand interactions are disrupted, the reduced binding affinity in the outward-facing conformation allows dissociation of the ligand from the binding cavity. Inward-facing and outward-facing conformations of the ABCB1 MDs are seen from the outside of the cell. Loops and the NBD are omitted for clarity. Residues involved in ligand binding are shown as sticks. Residues residing on TMHs 1, 6, 7 and 12 are shown in red. Residues contributed from other helices are rendered in green. Arrows refer to helix rotation from the inward to the outward state. To generate the outward-facing view, the residues from ABCB1 were superimposed onto the outward-facing structure of S. thyphimurium MsbA using Modeller. Figure was generated in Pymol.