title: Determining the dimensions of the drug-binding domain of human P-glycoprotein using thiol cross-linking compounds as molecular rulers.
abstract: The human multidrug resistance P-glycoprotein (P-gp) interacts with a broad range of compounds with diverse structures and sizes. There is considerable evidence indicating that residues in transmembrane segments 4-6 and 10-12 form the drug-binding site. We attempted to measure the size of the drug-binding site by using thiol-specific methanethiosulfonate (MTS) cross-linkers containing spacer arms of 2 to 17 atoms. The majority of these cross-linkers were also substrates of P-gp, because they stimulated ATPase activity (2.5- to 10.1-fold). 36 P-gp mutants with pairs of cysteine residues introduced into transmembrane segments 4-6 and 10-12 were analyzed after reaction with 0.2 mm MTS cross-linker at 4 degrees C. The cross-linked product migrated with lower mobility than native P-gp in SDS gels. 13 P-gp mutants were cross-linked by MTS cross-linkers with spacer arms of 9-25 A. Vinblastine and cyclosporin A inhibited cross-linking. The emerging picture from these results and other studies is that the drug-binding domain is large enough to accommodate compounds of different sizes and that the drug-binding domain is "funnel" shaped, narrow at the cytoplasmic side, at least 9-25 A in the middle, and wider still at the extracellular surface.
authors.affiliation: Department of Medicine, Canadian Institutes for Health Research Group in Membrane Biology, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: T W
authors.initials: TW
authors.lastname: Loo
authors.forename: D M
authors.initials: DM
authors.lastname: Clarke
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D003432:Cross-Linking Reagents / Q000378:metabolism*; D006801:Humans; D017434:Protein Structure, Tertiary; D013438:Sulfhydryl Compounds / Q000378:metabolism*
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D003432:Cross-Linking Reagents; D013438:Sulfhydryl Compounds