Allosteric modulation of the human P-glycoprotein involves conformational changes mimicking catalytic transition intermediates

• Published in: Archives of biochemistry and biophysics 2006-06, Vol.450 (1), p.100-112
• Main Authors: Ghosh, Pratiti, Moitra, Karobi, Maki, Nazli, Dey, Saibal
• Format: Article
• Language: English
• Subjects: ABCB1; Adenosine Triphosphate - analogs & derivatives; Adenosine Triphosphate - chemistry; Allosteric Regulation - genetics; Allosteric Site - genetics; Amino Acid Substitution; Animals; ATP-Binding Cassette, Sub-Family B, Member 1 - chemistry; ATP-Binding Cassette, Sub-Family B, Member 1 - genetics; ATP-Binding Cassette, Sub-Family B, Member 1 - metabolism; Biological Transport - drug effects; Biological Transport - genetics; Flupenthixol - chemistry; Flupentixol; Humans; Mice; N-α-Benzoylarginine- l- p-nitroanilide (BAPNA); NIH 3T3 Cells; Point Mutation; Protein Structure, Tertiary - drug effects; Protein Structure, Tertiary - genetics; Thioxanthene derivatives; Trypsin digestion; Vanadate-trapping; Vinblastine - chemistry
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2006.02.025

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 ATPγS, 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.