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J. Biol. Chem., Vol. 279, Issue 33, 34913-34921, August 13, 2004

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The Topography of Transmembrane Segment Six Is Altered during the Catalytic Cycle of P-glycoprotein^*

Alice Rothnie, Janet Storm, Jeff Campbell¶, Kenneth J. Linton||, Ian D. Kerr**, and Richard Callaghan

From the Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford, Oxford, OX3 9DU, ^¶Laboratory of Molecular Biophysics, University of Oxford, Rex Richards Bldg., South Parks Rd., Oxford OX1 3QU, ^||Medical Research Council Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital Campus, Du Cane Rd., London, W12 0NN, and ^**Centre for Biochemistry and Cell Biology, School of Biomedical Sciences, University of Nottingham, Queen's Medical Centre, Nottingham, NG7 2UH, United Kingdom

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'-(,-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.

Received for publication, May 13, 2004 , and in revised form, June 9, 2004.

^* This work was funded by Cancer Research United Kingdom Program Grant SP1861/0401. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a Medical Research Council Ph.D. studentship.

To whom correspondence should be addressed: Tel.: 44-1865-221-110; Fax: 44-1865-221-834; E-mail: richard.callaghan{at}ndcls.ox.ac.uk.

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