Drug-stimulated ATPase activity of human P-glycoprotein is blocked by disulfide cross-linking between the nucleotide-binding sites

P-glycoprotein (P-gp) is an ATP-dependent drug pump that contains two nucleotide-binding domains (NBDs). Disulfide cross-linking analysis was done to determine if the two NBDs are close to each other. Residues within or close to the Walker A (GNSGCGKS in NDB1 and GSSGCGKS in NBD2) sequences for nucleotide binding were replaced with cysteine, and the mutant P-gps were subjected to oxidative cross-linking. Cross-linking was detected in two mutants, G427C(NBD1)/Cys-1074(NBD2) and L439C(NBD1)/Cys-1074(NBD2), because the cross-linked proteins migrated slower in SDS gels. Mutants G427C(NBD1)/Cys-1074(NBD2) and L439C(NBD1)/Cys-1074(NBD2) retained 10% and 82%, respectively, of the drug-stimulated ATPase activity relative to that of Cys-less P-gp. The cross-linking properties of the more active mutant L439C(NBD1)/Cys-1074(NBD2) were then studied. Cross-linking was reversed by addition of dithiothreitol and could be prevented by pretreatment of the mutant with N-ethylmaleimide. Cross-linking was also inhibited by MgATP, but not by the verapamil. Oxidative cross-linking of mutant L439C(NBD1)/Cys-1074(NBD2) resulted in almost complete inhibition of drug-stimulated ATPase activity. More than 60% of the drug-stimulated ATPase activity, however, was recovered after treatment with dithiothreitol. The results indicate that the two predicted nucleotide-binding sites are close to each other and that cross-linking inhibits ATP hydrolysis.