Cross-linking of human multidrug resistance P-glycoprotein by the substrate, Tris-(2-maleimidoethyl)amine, is altered by ATP hydrolysis

We identified a thiol-reactive substrate, Tris-(-2-maleimidoethyl)amine (TM EA), to explore the contribution of the TM segments 6 and 12 of the human m ultidrug resistance P-glycoprotein (P-gp) during transport. TMEA is a trifu nctional maleimide and stimulated the ATPase activity of Cys-less P-gp abou t 7-fold. Cysteine-scanning mutagenesis of TM12 showed that the activity of mutant V982C was inhibited by TMEA. P-gp mutants containing V982C (TM12) a nd another cysteine in TM6 were constructed and tested for cross-linking wi th TAMA. A cross-linked product was observed in SDS-polyacrylamide gel elec trophoresis for mutant L339C(TM6)/V982C(TM12). Cross-linking by TMEA also i nhibited the ATPase activity of the mutant protein. Substrates such as cycl osporin A, vinblastine, colchicine, or verapamil inhibited cross-linking by TMEA. In the presence of ATP at 37 degreesC, cross-linking of mutant L339C /V982C was decreased. In contrast, there was enhanced cross-linking of muta nt F343C(TM6)/V982C(TM12) in the presence of ATP. These results show that c ross-linking must be within the drug-binding domain, that residues L339C(TM 6)/V982C(TM12) must be at least 10 Angstrom, apart, and that ATP hydrolysis promotes rotation of one or both TM helices.