Analysis of catalytic carboxylate mutants E552Q and E1197Q suggests asymmetric ATP hydrolysis by the two nucleotide-binding domains of P-glycoprotein.
In the nucleotide-binding domains (NBDs) of ABC transporters, such as mouse Mdr3 P-glycoprotein (P-gp), an invariant carboxylate residue (E552 in NBD1; E1197 in NBD2) immediately follows the Walker B motif (hyd(4)DE/D). Removal of the negative charge in mutants E552Q and E1197Q abolishes drug-stimulated ATPase activity measured by P(i) release. Surprisingly, drug-stimulated trapping of 8-azido-[alpha-(32)P]ATP is still observed in the mutants in both the presence and absence of the transition-state analogue vanadate (V(i)), and ADP can be recovered from the trapped enzymes. The E552Q and E1197Q mutants show characteristics similar to those of the wild-type (WT) enzyme with respect to 8-azido-[alpha-(32)P]ATP binding and 8-azido-[alpha-(32)P]nucleotide trapping, with the latter being both Mg(2+) and temperature dependent. Importantly, drug-stimulated nucleotide trapping in E552Q is stimulated by V(i) and resembles the WT enzyme, while it is almost completely V(i) insensitive in E1197Q. Similar nucleotide trapping properties are observed when aluminum fluoride or beryllium fluoride is used as an alternate transition-state analogue. Partial proteolytic cleavage of photolabeled enzymes indicates that, in the absence of V(i), nucleotide trapping occurs exclusively at the mutant NBD, whereas in the presence of V(i), nucleotide trapping occurs at both NBDs. Together, these results suggest that there is single-site turnover occurring in the E552Q and E1197Q mutants and that ADP release from the mutant site, or another catalytic step, is impaired in these mutants. Furthermore, our results support a model in which the two NBDs of P-gp are not functionally equivalent.
Department of Biochemistry and McGill Cancer Centre, McGill University, Montréal, Québec, Canada H3G 1Y6.
Isabelle
I
Carrier
Michel
M
Julien
Philippe
P
Gros
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000737:chemistry* / Q000235:genetics; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics; D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry*; D020816:Amino Acid Motifs / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002264:Carboxylic Acids / Q000737:chemistry*; D002384:Catalysis; D018432:Drug Resistance, Multiple / Q000235:genetics; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D005973:Glutamine / Q000235:genetics; D006868:Hydrolysis; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary / Q000235:genetics
D018435:ATP Binding Cassette Transporter, Subfamily B; D018528:ATP-Binding Cassette Transporters; D002264:Carboxylic Acids; D047908:Intracellular Signaling Peptides and Proteins; C400289:NUBP2 protein, mouse; C400290:Nubp1 protein, mouse; D005973:Glutamine; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; C095122:multidrug resistance protein 3; D000251:Adenosine Triphosphatases; D019204:GTP-Binding Proteins