Mutational analysis of conserved carboxylate residues in the nucleotide binding sites of P-glycoprotein

Mutagenesis was used to investigate the functional role of six pairs of asp artate and glutamate residues (D450/D1093, E482/E1125, E552/E1197, D558/D12 03, D592/D1237, and E604/E1249) that are highly conserved in the nucleotide binding sites of P-glycoprotein (Mdr3) and of other ABC transporters. Remo val of the charge in E552Q/E1197Q and D558N/D1203N produced proteins with s everely impaired biological activity when the proteins were analyzed in yea st cells for cellular resistance to FK506 and restoration of mating in a st e6 Delta mutant. Mutations at other acidic residues had no apparent effect in the same assays. These four mutants were expressed in Pichia pastoris, p urified to homogeneity, and biochemically characterized with respect to ATP ase activity. Studies with purified proteins showed that mutants D558N and D1203N retained 14 and 30% of the drug-stimulated ATPase activity of wild-t ype (WT) Mdr3, respectively, and vanadate trapping of 8-azido[alpha-P-32]nu cleotide confirmed slower basal and drug-stimulated X-azido-ATP hydrolysis compared to that for WT Mdr3, The E552Q and E1197Q mutants showed no drug-s timulated ATPase activity. Surprisingly, drugs did stimulate vanadate trapp ing of 8-azido[alpha-P-32]nucleotide in E552Q and E1197Q at a level similar to that of WT Mdr3. This suggests that formation of the catalytic transiti on state can occur in these mutants, and that the bond between the beta- an d gamma -phosphates is hydrolyzed, In addition, photolabeling by 8-azido[al pha-P-32]nucleotide in the presence or absence of drug was also detected in the absence of vanadate in these mutants. These results suggest that steps after the transition state, possibly involved in release of MgADP, are sev erely impaired in these mutant enzymes.