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 he 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 Leu(339), Ala(342), Leu(975), Val(982), and Ala(985) 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.