title: Substrate-induced conformational changes in the transmembrane segments of human P-glycoprotein. Direct evidence for the substrate-induced fit mechanism for drug binding.
abstract: The human multidrug resistance P-glycoprotein (P-gp, ABCB1) is quite promiscuous in that it can transport a broad range of structurally diverse compounds out of the cell. We hypothesized that the transmembrane (TM) segments that constitute the drug-binding site are quite mobile such that drug binding occurs through a "substrate-induced fit" mechanism. Here, we used cysteine-scanning mutagenesis and oxidative cross-linking to test for substrate-induced changes in the TM segments. Pairs of cysteines were introduced into a Cys-less P-gp and the mutants treated with oxidant (copper phenanthroline) in the presence or absence of various drug substrates. We show that cyclosporin A promoted cross-linking between residues P350C(TM6)/G939C(TM11), while colchicine and demecolcine promoted cross-linking between residues P350C(TM6)/V991C(TM12). Progesterone promoted cross-linking between residues P350C(TM6)/A935C(TM11), P350C(TM6)/G939C(TM11), as well as between residues P350C(TM6)/V991C(TM12). Other substrates such as vinblastine, verapamil, cis-(Z)-flupenthixol or trans-(E)-flupenthixol did not induce cross-linking at these sites. These results provide direct evidence that the packing of the TM segments in the drug-binding site is changed when P-gp binds to a particular substrate. The induced-fit mechanism explains how P-gp can accommodate a broad range of compounds.
authors.affiliation: Canadian Institutes of Health Research Group in Membrane Biology, Department of Medicine, University of Toronto, Ontario M5S 1A8, Canada.
authors.forename: Tip W
authors.initials: TW
authors.lastname: Loo
authors.forename: M Claire
authors.initials: MC
authors.lastname: Bartlett
authors.forename: David M
authors.initials: DM
authors.lastname: Clarke
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003078:Colchicine / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000494:pharmacology; D016572:Cyclosporine / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D003703:Demecolcine / Q000494:pharmacology; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000737:chemistry; D006801:Humans; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011374:Progesterone / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity; D013997:Time Factors; D014162:Transfection
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D004220:Disulfides; D011374:Progesterone; D006639:Histidine; D016572:Cyclosporine; D000251:Adenosine Triphosphatases; D003078:Colchicine; D003703:Demecolcine