Identification of residues in the drug-binding site of human P-glycoprotein using a thiol-reactive substrate.
We identified a thiol-reactive compound, dibromobimane (dBBn), that was a potent stimulator (8.2-fold) of the ATPase activity of Cys-less P-glycoprotein. We then used this compound together with cysteine-scanning mutagenesis to identify residues in transmembrane segment (TM) 6 and TM12 that are important for function. TM6 and TM12 lie close to each other in the tertiary structure and are postulated to be important for drug-protein interactions. The majority of P-glycoprotein mutants containing a single cysteine residue retained substantial amounts of drug-stimulated ATPase activity and were not inhibited by dBBn. The ATPase activities of mutants L339C, A342C, L975C, V982C, and A985C, however, were markedly inhibited (>60%) by dBBn. The drug substrates verapamil, vinblastine, and colchicine protected these mutants against inhibition by dBBn, suggesting that these residues are important for interaction of substrates with P-glycoprotein. We previously showed that residues Leu339, Ala342, Leu975, Val982, and Ala985 lie along the point of contact between helices TM6 and TM12, when both are aligned in a left-handed coiled coil (Loo, T. W., and Clarke, D. M. (1997) J. Biol. Chem. 272, 20986-20989). Taken together, these results suggest that the interface between TM6 and TM12 likely forms part of the potential drug-binding pocket in P-glycoprotein.
Loo
T W
TW
Medical Research Council Group in Membrane Biology, Departments of Medicine and Biochemistry, University of Toronto, Ontario M5S 1A8, Canada.
Clarke
D M
DM
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D001643:Bridged Bicyclo Compounds / Q000494:pharmacology; D003078:Colchicine / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D016297:Mutagenesis, Site-Directed; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D014700:Verapamil / Q000378:metabolism; D014747:Vinblastine / Q000378:metabolism
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001643:Bridged Bicyclo Compounds; D004791:Enzyme Inhibitors; D013438:Sulfhydryl Compounds; D014747:Vinblastine; C037094:dibromobimane; D014700:Verapamil; D000251:Adenosine Triphosphatases; D003545:Cysteine; D003078:Colchicine