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Papers In Press, published online ahead of print June 10, 2004
Nuffield Department of Clinical Laboratory Sciences, University of Oxford, Oxford, Oxfordshire OX3 9DU
Corresponding Author: richard.callaghan{at}ndcls.ox.ac.uk
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 (CM) readily labelled all introduced cysteine residues, whereas the hydrophilic fluorescein maleimide (FM) only labelled residue 343C. The amphiphilic BODIPY-maleimide (BM) displayed a more complex labelling profile. The extent of labelling with CM did not vary during the catalytic cycle, whilst FM labelling of F343C was lost following nucleotide binding or hydrolysis. BM labelling was markedly altered during the catalytic cycle and indicated that the AMP-PNP 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.
J. Biol. Chem, 10.1074/jbc.M405336200
Submitted on May 13, 2004
Revised on June 9, 2004
Accepted on June 10, 2004
The topography of transmembrane segment six is altered during the catalytic cycle of P-glycoprotein
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