title: Multiple transport-active binding sites are available for a single substrate on human P-glycoprotein (ABCB1).
abstract: P-glycoprotein (Pgp, ABCB1) is an ATP-Binding Cassette (ABC) transporter that is associated with the development of multidrug resistance in cancer cells. Pgp transports a variety of chemically dissimilar amphipathic compounds using the energy from ATP hydrolysis. In the present study, to elucidate the binding sites on Pgp for substrates and modulators, we employed site-directed mutagenesis, cell- and membrane-based assays, molecular modeling and docking. We generated single, double and triple mutants with substitutions of the Y307, F343, Q725, F728, F978 and V982 residues at the proposed drug-binding site with cys in a cysless Pgp, and expressed them in insect and mammalian cells using a baculovirus expression system. All the mutant proteins were expressed at the cell surface to the same extent as the cysless wild-type Pgp. With substitution of three residues of the pocket (Y307, Q725 and V982) with cysteine in a cysless Pgp, QZ59S-SSS, cyclosporine A, tariquidar, valinomycin and FSBA lose the ability to inhibit the labeling of Pgp with a transport substrate, [(125)I]-Iodoarylazidoprazosin, indicating these drugs cannot bind at their primary binding sites. However, the drugs can modulate the ATP hydrolysis of the mutant Pgps, demonstrating that they bind at secondary sites. In addition, the transport of six fluorescent substrates in HeLa cells expressing triple mutant (Y307C/Q725C/V982C) Pgp is also not significantly altered, showing that substrates bound at secondary sites are still transported. The homology modeling of human Pgp and substrate and modulator docking studies support the biochemical and transport data. In aggregate, our results demonstrate that a large flexible pocket in the Pgp transmembrane domains is able to bind chemically diverse compounds. When residues of the primary drug-binding site are mutated, substrates and modulators bind to secondary sites on the transporter and more than one transport-active binding site is available for each substrate.
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Eduardo E
authors.initials: EE
authors.lastname: Chufan
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Khyati
authors.initials: K
authors.lastname: Kapoor
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Hong-May
authors.initials: HM
authors.lastname: Sim
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John's University, Queens, New York, United States of America.
authors.forename: Satyakam
authors.initials: S
authors.lastname: Singh
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John's University, Queens, New York, United States of America.
authors.forename: Tanaji T
authors.initials: TT
authors.lastname: Talele
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Stewart R
authors.initials: SR
authors.lastname: Durell
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Suresh V
authors.initials: SV
authors.lastname: Ambudkar
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D045744:Cell Line, Tumor; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D014161:Transduction, Genetic
chemical_list: C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D005456:Fluorescent Dyes; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases