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Structural evidence has demonstrated that P-glycoprotein (P-gp) undergoes considerable conformational changes during catalysis, and these alterations are important in drug interaction. Knowledge of which regions in P-gp undergo conformational alterations will provide vital information to elucidate the locations of drug binding sites and the mechanism of coupling. A number of investigations have implicated transmembrane segment six (TM6) in drug-P-gp interactions, and a cysteine-scanning mutagenesis approach was directed to this segment. Introduction of cysteine residues into TM6 did not disturb basal or drug-stimulated ATPase activity per se. Under basal conditions the hydrophobic probe coumarin maleimide readily labeled all introduced cysteine residues, whereas the hydrophilic fluorescein maleimide only labeled residue Cys-343. The amphiphilic BODIPY-maleimide displayed a more complex labeling profile. The extent of labeling with coumarin maleimide did not vary during the catalytic cycle, whereas fluorescein maleimide labeling of F343C was lost after nucleotide binding or hydrolysis. BODIPY-maleimide labeling was markedly altered during the catalytic cycle and indicated that the adenosine 5'-(beta,gamma-imino)triphosphate-bound and ADP/vanadate-trapped intermediates were conformationally distinct. Our data are reconciled with a recent atomic scale model of P-gp and are consistent with a tilting of TM6 in response to nucleotide binding and ATP hydrolysis.

Original publication

DOI

10.1074/jbc.M405336200

Type

Journal article

Journal

J Biol Chem

Publication Date

13/08/2004

Volume

279

Pages

34913 - 34921

Keywords

ATP-Binding Cassette, Sub-Family B, Member 1, Adenosine Triphosphatases, Adenosine Triphosphate, Animals, Binding Sites, Boron Compounds, Catalysis, Cell Line, Cell Membrane, Codon, Coumarins, Cysteine, Electrophoresis, Polyacrylamide Gel, Hydrolysis, Insecta, Kinetics, Maleimides, Models, Chemical, Models, Molecular, Protein Conformation, Protein Isoforms, Protein Structure, Tertiary, Recombinant Proteins, Sequence Analysis, DNA