title: Allosteric modulation of the human P-glycoprotein involves conformational changes mimicking catalytic transition intermediates.
abstract: The drug transport function of human P-glycoprotein (Pgp, ABCB1) can be inhibited by a number of pharmacological agents collectively referred to as modulators or reversing agents. In this study, we demonstrate that certain thioxanthene-based Pgp modulators with an allosteric mode of action induce a distinct conformational change in the cytosolic domain of Pgp, which alters susceptibility to proteolytic digestion. Both cis and trans-isomers of the Pgp modulator flupentixol confer considerable protection of an 80 kDa Pgp fragment against trypsin digestion, that is recognized by a polyclonal antibody specific for the NH(2)-terminal half to Pgp. The protection by flupentixol is abolished in the Pgp F983A mutant that is impaired in modulation by flupentixols, indicating involvement of the allosteric site in generating the conformational change. A similar protection to an 80 kDa fragment is conferred by ATP, its nonhydrolyzable analog ATPgammaS, and by trapping of ADP-vanadate at the catalytic domain, but not by transport substrate vinblastine or by the competitive modulator cyclosporin A, suggesting different outcomes from modulator interaction at the allosteric site and at the substrate site. In summary, we demonstrate that allosteric interaction of flupentixols with Pgp generates conformational changes that mimic catalytic transition intermediates induced by nucleotide binding and hydrolysis, which may play a crucial role in allosteric inhibition of Pgp-mediated drug transport.
authors.affiliation: Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, F. Edward Hébert School of Medicine, 4301 Jones Bridge Road, Bethesda, MD 20814-4799, USA.
authors.forename: Pratiti
authors.initials: P
authors.lastname: Ghosh
authors.forename: Karobi
authors.initials: K
authors.lastname: Moitra
authors.forename: Nazli
authors.initials: N
authors.lastname: Maki
authors.forename: Saibal
authors.initials: S
authors.lastname: Dey
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D000494:Allosteric Regulation / Q000235:genetics; D000495:Allosteric Site / Q000235:genetics; D019943:Amino Acid Substitution; D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000235:genetics; D005475:Flupenthixol / Q000737:chemistry*; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D017354:Point Mutation; D017434:Protein Structure, Tertiary / Q000187:drug effects / Q000235:genetics; D014747:Vinblastine / Q000737:chemistry
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D014747:Vinblastine; D000255:Adenosine Triphosphate; D005475:Flupenthixol