Covalent modification of human P-glycoprotein mutants containing a single cysteine in either nucleotide-binding fold abolishes drug-stimulated ATPase activity.

The ATPase activity of P-glycoprotein is inactivated by N-ethylmaleimide (NEM), which is postulated to modify cysteine residues within either of the homology A consensus sequences for nucleotide binding (GNSGCGKS and GSSGCGKS, respectively) (Al-Shawi, M. K., Urbatsch, I. L., and Senior, A. E. (1994) J. Biol. Chem. 269, 8986-8992). To test this postulate as well as determine the contribution of either nucleotide-binding domain to function, a Cys-less mutant was constructed, and then a single cysteine residue was reintroduced back into each nucleotide-binding consensus sequence. We then tested the sensitivity of the ATPase activity of each mutant to covalent modification by NEM. It was found that covalent modification of a single cysteine residue within either nucleotide-binding consensus sequence (Cys-431 and Cys-1074, respectively) with NEM inhibited drug-stimulated ATPase activity of P-glycoprotein. The concentrations of NEM required for half-maximal inactivation of ATPase activity were 7 and 35 microM for mutants Cys-431 and Cys-1074, respectively. In both cases, inactivation of ATPase activity by NEM was prevented by ATP. These results suggest that both nucleotide-binding domains may need to bind ATP to couple drug binding to ATPase activity.