title: Cysteines introduced into extracellular loops 1 and 4 of human P-glycoprotein that are close only in the open conformation spontaneously form a disulfide bond that inhibits drug efflux and ATPase activity.
abstract: P-glycoprotein (P-gp) is an ATP-binding cassette drug pump that protects us from toxic compounds and confers multidrug resistance. The protein is organized into two halves. The halves contain a transmembrane domain (TMD) with six transmembrane segments and a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the TMD1/TMD2 and NBD1/NBD2 interfaces, respectively. ATP-dependent drug efflux involves changes between the open inward-facing (NBDs apart, extracellular loops (ECLs) close together) and the closed outward-facing (NBDs close together, ECLs apart) conformations. It is controversial, however, whether the open conformation only exists transiently in intact cells because of the presence of high levels of ATP. To test for the presence of an open conformation in intact cells, reporter cysteines were placed in extracellular loops 1 (A80C, N half) and 4 (R741C, C half). The rationale was that cysteines A80C/R741C would only come close enough to form a disulfide bond in an open conformation (6.9 Å apart) because they are separated widely (30.4 Å apart) in the closed conformation. It was observed that the mutant A80C/R741C cross-linked spontaneously (>90%) when expressed in cells. In contrast to previous reports showing that trapping P-gp in a closed conformation highly activated ATPase activity, here we show that A80C/R741C cross-linking inhibited ATPase activity and drug efflux. Both activities were restored when the cross-linked mutant was treated with a thiol-reducing agent. The results show that an open conformation can be readily detected in cells and that cross-linking of cysteines placed in ECLs 1 and 4 inhibits activity.
authors: From the Departments of Medicine and Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors: Tip W
authors: TW
authors: Loo
authors: From the Departments of Medicine and Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada david.clarke@utoronto.ca.
authors: David M
authors: DM
authors: Clarke
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D018432:Drug Resistance, Multiple / Q000187:drug effects; D006026:Glycoside Hydrolases / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D006868:Hydrolysis; D015151:Immunoblotting; D008958:Models, Molecular; D009154:Mutation; D004364:Pharmaceutical Preparations / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D004220:Disulfides; D004364:Pharmaceutical Preparations; D000255:Adenosine Triphosphate; D006026:Glycoside Hydrolases; D000251:Adenosine Triphosphatases; D003545:Cysteine; D004229:Dithiothreitol
keywords: ABC Transporter; Drug Resistance; Membrane Enzyme; Membrane Protein; Protein Cross-linking