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 aspartate and glutamate residues (D450/D1093, E482/E1125, E552/E1197, D558/D1203, D592/D1237, and E604/E1249) that are highly conserved in the nucleotide binding sites of P-glycoprotein (Mdr3) and of other ABC transporters. Removal of the charge in E552Q/E1197Q and D558N/D1203N produced proteins with severely impaired biological activity when the proteins were analyzed in yeast cells for cellular resistance to FK506 and restoration of mating in a ste6Delta mutant. Mutations at other acidic residues had no apparent effect in the same assays. These four mutants were expressed in Pichia pastoris, purified to homogeneity, and biochemically characterized with respect to ATPase activity. Studies with purified proteins showed that mutants D558N and D1203N retained 14 and 30% of the drug-stimulated ATPase activity of wild-type (WT) Mdr3, respectively, and vanadate trapping of 8-azido[alpha-(32)P]nucleotide confirmed slower basal and drug-stimulated 8-azido-ATP hydrolysis compared to that for WT Mdr3. The E552Q and E1197Q mutants showed no drug-stimulated ATPase activity. Surprisingly, drugs did stimulate vanadate trapping of 8-azido[alpha-(32)P]nucleotide in E552Q and E1197Q at a level similar to that of WT Mdr3. This suggests that formation of the catalytic transition state can occur in these mutants, and that the bond between the beta- and gamma-phosphates is hydrolyzed. In addition, photolabeling by 8-azido[alpha-(32)P]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 severely impaired in these mutant enzymes.
Urbatsch
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Department of Biochemistry, McGill University, Montréal, Québec, Canada H3G 1Y6.
Julien
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Carrier
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Rousseau
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ME
Cayrol
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Gros
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D018435:ATP Binding Cassette Transporter, Subfamily B / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001386:Azides / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002264:Carboxylic Acids / Q000378:metabolism*; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D018432:Drug Resistance, Multiple; D005822:Genetic Vectors / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000378:metabolism*; D019920:Photoaffinity Labels / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D014638:Vanadates / Q000378:metabolism
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D001386:Azides; D002264:Carboxylic Acids; D009841:Oligonucleotides; D019920:Photoaffinity Labels; D014638:Vanadates; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; C095122:multidrug resistance protein 3