title: Location of the rhodamine-binding site in the human multidrug resistance P-glycoprotein.
abstract: The human multidrug resistance P-glycoprotein (P-gp) pumps a wide variety of structurally diverse compounds out of the cell. It is an ATP-binding cassette transporter with two nucleotide-binding domains and two transmembrane (TM) domains. One class of compounds transported by P-gp is the rhodamine dyes. A P-gp deletion mutant (residues 1-379 plus 681-1025) with only the TM domains retained the ability to bind rhodamine. Therefore, to identify the residues involved in rhodamine binding, 252 mutants containing a cysteine in the predicted TM segments were generated and reacted with a thiol-reactive analog of rhodamine, methanethiosulfonate (MTS)-rhodamine. The activities of 28 mutants (in TMs 2-12) were inhibited by at least 50% after reaction with MTS-rhodamine. The activities of five mutants, I340C(TM6), A841C(TM9), L975C(TM12), V981C(TM12), and V982C(TM12), however, were significantly protected from inhibition by MTS-rhodamine by pretreatment with rhodamine B, indicating that residues in TMs 6, 9, and 12 contribute to the binding of rhodamine dyes. These results, together with those from previous labeling studies with other thiol-reactive compounds, dibromobimane, MTS-verapamil, and MTS-cross-linker substrates, indicate that common residues are involved in the binding of structurally different drug substrates and that P-gp has a common drug-binding site. The results support the "substrate-induced fit" hypothesis for drug binding.
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: David M
authors.initials: DM
authors.lastname: Clarke
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000737:chemistry* / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D001665:Binding Sites; D001643:Bridged Bicyclo Compounds / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000494:pharmacology; D006801:Humans; D008956:Models, Chemical; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry* / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012235:Rhodamines / Q000378:metabolism* / Q000494:pharmacology; D014162:Transfection; D014700:Verapamil / Q000494:pharmacology
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D000251:Adenosine Triphosphatases; D001643:Bridged Bicyclo Compounds; D002121:Calcium Channel Blockers; D003545:Cysteine; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D012235:Rhodamines; D014700:Verapamil; C037094:dibromobimane; C029773:rhodamine B; C421109:multidrug resistance-associated protein 1