Synergy between conserved ABC signature Ser residues in P-glycoprotein catalysis.
Functional roles of the two ABC signature sequences ("LSGGQ") in the N- and C-terminal nucleotide binding domains of P-glycoprotein were studied by mutating the conserved Ser residues to Ala. The two single mutants (S528A; S1173A) each impaired ATPase activity mildly, and showed generally symmetrical effects on function, consistent with equivalent mechanistic roles of the two nucleotide sites. Synergy between the two mutations when combined was remarkable and resulted in strong catalytic impairment. The Ser residues are not involved significantly in MgATP- or MgADP-binding or in interdomain communication between catalytic sites and drug binding sites. Retention of product MgADP is not the cause of reduced turnover. Mutation of Ser to Ala reduced the strength of interaction with the chemical transition state specifically, as shown by vanadate-ADP and beryllium fluoride-ADP trapping experiments. Therefore, the two conserved ABC signature motif Ser residues of P-glycoprotein cooperatively accelerate ATP hydrolysis via chemical transition state interaction. Because the transition state complex is currently believed to form in the dimerized state of the nucleotide binding domains, one may also conclude that both Ser-OH are necessary for correct formation of the dimer state.
Tombline
Gregory
G
Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642, USA.
Bartholomew
Lori
L
Gimi
Khursheed
K
Tyndall
Grace A
GA
Senior
Alan E
AE
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000235:genetics*; D000244:Adenosine Diphosphate / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000409:Alanine / Q000737:chemistry; D020816:Amino Acid Motifs; D000818:Animals; D001608:Beryllium / Q000494:pharmacology; D001665:Binding Sites; D002384:Catalysis; D002606:Charcoal / Q000494:pharmacology; D019281:Dimerization; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005459:Fluorides / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008055:Lipids / Q000737:chemistry; D051379:Mice; D016296:Mutagenesis; D009154:Mutation; D010843:Pichia / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry; D013696:Temperature; D013997:Time Factors; D014638:Vanadates / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D008024:Ligands; D008055:Lipids; D002606:Charcoal; D014638:Vanadates; D012694:Serine; C035982:beryllium fluoride; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000409:Alanine; D001608:Beryllium; D005459:Fluorides; D004229:Dithiothreitol