Identifying the location of the drug binding site(s) in P-glycoprotein

Abstract

P-glycoprotein (P-gp), an ABC transporter protein, is characterized by its ability to recognize, bind, and efflux over 300 chemically and structurally unrelated compounds. P-gp overexpression in cancer cells confers multidrug resistance (MDR) by preventing sufficient accumulation of anticancer drugs within the cell, thus avoiding their cytotoxic effects. P-gp harnesses the energy from ATP hydrolysis to translocate substrates across the plasma membrane against a concentration gradient. Biochemical and structural investigations have identified the presence of a large aqueous central cavity which is likely the location for the drug binding sites (DBSs). The substrate polyspecificity displayed by P-gp is imparted by the existence of at least four pharmacologically distinct DBSs located within the transmembrane domain (TMD). However, whether these sites are spatially distinct from each other was not clear. The current investigation has identified several key contact residues of four pharmacologically distinct substrates/modulators of P-gp (nicardipine, vinblastine, rhodamine 123, and paclitaxel), known to bind at the four identified sites. Seven residues in various TMDs were mutated to a cysteine. Biochemical assays using purified, reconstituted P-gp expressing each mutant isoform was used to identify which contact residues were implicated in the binding of each drug. For rhodamine 123 binding, the identified contact residues were located within the central cavity of P-gp. However, for vinblastine, paclitaxel, and nicardipine, the implicated contact residues were located at the lipid-protein interface rather than the central cavity. A key residue (F978) located within the central cavity is believed to be involved in the interdomain communication between the TMDs and the nucleotide binding domains (NBD; site of ATP hydrolysis). Collectively, data presented here suggests the existence of at least four spatially distinct drug binding sites that are connected by a single translocation pore in the central cavity.