Mechanics and pharmacology of substrate selection and transport by eukaryotic ABC exporters

Abstract

Much structural information has been amassed on ATP-binding cassette (ABC) transporters, including hundreds of structures of isolated domains and an increasing array of full-length transporters. The structures capture different steps in the transport cycle and have aided in the design and interpretation of computational simulations and biophysics experiments. These data provide a maturing, although still incomplete, elucidation of the protein dynamics and mechanisms of substrate selection and transit through the transporters. We present an updated view of the classical alternating-access mechanism as it applies to eukaryotic ABC transporters, focusing on type I exporters. Our model helps frame the progress in, and remaining questions about, transporter energetics, how substrates are selected and how ATP is consumed to perform work at the molecular scale. Many human ABC transporters are associated with disease; we highlight progress in understanding their pharmacology through the lens of structural biology and describe how this knowledge suggests approaches to pharmacologically targeting these transporters.

Fig. 1.. Structural similarities in ABCB, ABCC and ABCD exporters in contrast to ABCA and ABCG transporters.

(a) Schematic structures of type I (ABCB, ABCC and ABCD; left) and type II (ABCA and ABCG; right) exporters with the core domain consisting of pseudo-symmetric TMDs (dark and light grey) each coupled to an NBD (dark and light yellow) where Mg²⁺-ATP (orange and black) bind. Some transporters contain an extra TMD0 (lilac) that can modulate the transporter. (b) Representative type I ABC exporter structures loosely binned to represent conformational states in the consensus transport cycle. Inward-open: LTC4-bound bovine MRP1 bound (PDB 5UJA); C. elegans P-gp (PDB 4F4C); C. jejuni PglK (PDB 5C76); AMPPNP-bound human ABCB10 (PDB 4AYX). Outward-occluded: Zosuquidar-bound human-mouse chimeric P-gp (UIC2 Fab not shown) (PDB 6FN1); ATP-bound E. coli McjD (PDB 5OFR). Outward-open: ATP-bound hydrolysis-deficient bovine MRP1 (PDB 6BHU); ATP-bound hydrolysis-deficient human P-gp (PDB 6C0V). Inward-occluded: E. coli McjD (PDB 5OFP). (c) Structures of NBDs during a transport and ATPase cycle viewed from the cytosol looking at the membrane. Clockwise from top left: Separated apo-NBDs in inward-open state (PDB 4F4C); Interacting Mg²⁺-ATP-bound NBDs in outward-occluded state (PDB 5OFR); ATPase-competent Mg²⁺-AMPPNP-bound NBD dimer in outward-open state (PDB 2ONJ); Interacting NBDs in inward-occluded state after ADP and phosphate release (PDB 5OFP). (d) Structures of stereotypical type II exporters: nucleotide-free inward-open ABCG5/G8 (left; PDB 5DO7), inward-open ABCG2 with substrate estrone-3-sulfate (middle, PDB 6HCO), and ATP-bound outward-open ABCG2 (right; PDB 6HBU).

Fig. 2.. Experimentally identified substrate-interacting residues suggest binding site diversity among ABC exporters.

(a) Biochemically identified substrate-interacting residues in TAP and P-gp. Left: Zosuquidar-bound P-gp (UIC2 Fab not shown; PDB 6FN1). Purple spheres represent substrate-interacting residues from structure and crosslinking experiments. Right: ICP47-bound TAP (PDB 5U1D). Pink segments crosslinked to substrate peptides. Purple spheres represent crosslinked residues, variants that affect substrate transport and selectivity, and substrate-docking studies. (b) Substrate-bound structures highlight the chemical diversity of interactions distributed across the TMD cavity. Left: LPS-bound E. coli MsbA (PDB 5TV4). Right: LTC4-bound bovine MRP1 (PDB 5UJA). Pink and purple spheres represent substrate-interacting residues forming hydrophobic and polar contacts respectively. (c) TAP and P-gp substrate-interacting residues highlighted in (a) and (b) (purple) mapped onto the TAP TMD cavity viewed from the cytosol (PDB 5U1D). Cyan cloud highlights the combined positions of substrates LTC4 (PDB 5UJA) and Zosuquidar (PDB 6FN1). (d) Substrate-induced changes in TM4 and TM4’/10 with aligned structures of PBDE-100-bound P-gp (gray, PDB 4XWK), apo-P-gp (yellow, PDB 4F4C), Zosuquidar-bound P-gp (blue, PDB 6QEE), Taxol-bound P-gp (green, PDB 6QEX), QZ-Ala-bound P-gp (magenta, PDB 4Q9I), with the corresponding ligands as dotted surfaces. See Supplementary Table 1 for residue lists and references.

Fig. 3.. Consensus thermodynamic model for transport by ABC exporters.

Vertically aligned (a) energy diagram and (b) conformational state schematics for transport cycle of type I ABC exporters. The resting state (1) is inward open. Substrate and ATP binding promotes transition through substrate-bound occluded states (2, 3), to a locally stable outward-open state (4), with an ATP-dependent NBD dimer. Substrate-bound transporters have a lower transition energy (red box), accounting for the observed substrate stimulation. After substrate release (5), ATP hydrolysis destabilizes the NBD dimer and outward-open state, leading the transporter to transition through an occluded state (6) and ending at the inward-open state (7) concomitant with release nucleotide, resetting for another round of transport.

Fig. 4.. Structural insights into ABC transporter pharmacology.

(a) Structures of type I exporters bound to competitive inhibitors. From left: the core domains of human TAP bound to ICP47 (PDB 5U1D); chimeric mouse-human P-gp bound to two Zosuquidar molecules, a small-molecule inhibitor (PDB 6QEE); mouse P-gp bound to the marine pollutant PBDE-100 (PDB 4XWK); mouse P-gp bound to two QZ-Ala cyclopeptides (PDB 4Q9I), which may be a competing substrate. (b) Structure of type I exporters bound to non-competitive inhibitors. From left: C. merolae P-gp homodimer bound to two aCAP cyclopeptides (PDB 3WMG); C. jejuni PglK homodimer bound to a single Nb87 nanobody (PDB 5NBD); E. coli MsbA homodimer bound to LPS substrate (black) and two G907 small-molecule inhibitors (PDB 6BPL); chimeric mouse-human P-gp bound to the UIC2 antibody (only the Fv fragment is illustrated and the transporter is rotated ~180° relative to other panels to better view the interface; PDB 6FN4). Color-coding as in Fig. 1, with the inhibitors illustrated as cyan dotted surfaces.