title: Conformational analysis of human ATP-binding cassette transporter ABCB1 in lipid nanodiscs and inhibition by the antibodies MRK16 and UIC2.
abstract: The human ATP-binding cassette (ABC) transporter, P-glycoprotein (P-gp; ABCB1), mediates the ATP-dependent efflux of a variety of drugs. As a result, P-gp plays a critical role in tumor cell drug resistance and the pharmacokinetic properties of most drugs. P-gp exhibits extraordinary substrate and inhibitor promiscuity, resulting in a wide range of possible drug-drug interactions. Inhibitory antibodies have long been considered as a possible strategy to modulate P-gp-dependent cancer cell drug resistance, and it is widely suggested that the antibodies MRK16 and UIC2 inhibit P-gp by capturing a single isoform and preventing flux through the catalytic cycle. Although the crystal structures of many bacterial whole transporters, as well as isolated nucleotide-binding domains, have been solved, high resolution structural data for mammalian ABC transporters are currently lacking. It has been extremely difficult to determine the detailed mechanism of transport of P-gp, in part because it is difficult to obtain purified protein in well defined lipid systems. Here we exploit surface plasmon resonance (SPR) to probe conformational changes associated with these intermediate states for P-gp in lipid bilayer nanodiscs. The results indicate that P-gp in nanodiscs undergoes functionally relevant ligand-dependent conformational changes and that previously described inhibitory antibodies bind to multiple nucleotide-bound states but not the ADP-VO(4)-trapped state, which mimics the post-hydrolysis state. The results also suggest that the substrate drug vinblastine is released at stages that precede or follow the post-hydrolysis ADP-PO(4)·P-gp complex.
authors.affiliation: Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195-7610, USA.
authors.forename: Tasha K
authors.initials: TK
authors.lastname: Ritchie
authors.forename: Hyewon
authors.initials: H
authors.lastname: Kwon
authors.forename: William M
authors.initials: WM
authors.lastname: Atkins
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry*; D000972:Antineoplastic Agents, Phytogenic / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology; D001693:Biological Transport, Active / Q000502:physiology; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D014747:Vinblastine / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology
chemical_list: C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D058846:Antibodies, Monoclonal, Murine-Derived; D000972:Antineoplastic Agents, Phytogenic; D008051:Lipid Bilayers; D014747:Vinblastine; D000244:Adenosine Diphosphate