The human multidrug resistance P-glycoprotein (P-gp, ABCB1) uses ATP to transport many structurally diverse compounds out of the cell. It is an ABC transporter with two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). Recently, we showed that the "LSGGQ" motif in one NBD ((531)LSGGQ(535) in NBD1; (1176)LSGGQ(1180) in NBD2) is adjacent to the "Walker A" sequence ((1070)GSSGCGKS(1077) in NBD2; (427)GNSGCGKS(434) in NBD1) in the other NBD (Loo, T. W., Bartlett, M. C., and Clarke, D. M. (2002) J. Biol. Chem. 277, 41303-41306). Drug substrates can stimulate or inhibit the ATPase activity of P-gp. Here, we report the effect of drug binding on cross-linking between the LSGGQ signature and Walker A sites (Cys(431)(NBD1)/C1176C(NBD2) and Cys(1074)(NBD2)/L531C(NBD1), respectively). Seven drug substrates (calcein-AM, demecolcine, cis(Z)-flupentixol, verapamil, cyclosporin A, Hoechst 33342, and trans(E)-flupentixol) were tested for their effect on oxidative cross-linking. Substrates that stimulated the ATPase activity of P-gp (calcein-AM, demecolcine, cis(Z)-flupentixol, and verapamil) increased the rate of cross-linking between Cys(431)(NBD1-Walker A)/C1176C(NBD2-LSGGQ) and between Cys(1074)(NBD2-Walker A)/L531C(NBD1-LSGGQ) when compared with cross-linking in the absence of drug substrate. By contrast, substrates that inhibited ATPase activity (cyclosporin A, Hoechst 33342, and trans(E)-flupentixol) decreased the rate of cross-linking. These results indicate that interaction between the LSGGQ motifs and Walker A sites must be essential for coupling drug binding to ATP hydrolysis. Drug binding in the transmembrane domains can induce long range conformational changes in the NBDs, such that compounds that stimulate or inhibit ATPase activity must decrease and increase, respectively, the distance between the Walker A and LSGGQ sequences.