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J Biol Chem, Vol. 275, Issue 8, 5253-5256, February 25, 2000
From the Medical Research Council Group in Membrane Biology,
Department of Medicine and Department of Biochemistry, University of
Toronto, Toronto, Ontario M5S 1A8, Canada
Residues from several transmembrane (TM) segments
of P-glycoprotein (P-gp) likely form the drug-binding site(s). To
determine the organization of the TM segments, pairs of cysteine
residues were introduced into the predicted TM segments of a Cys-less
P-gp, and the mutant protein was subjected to oxidative cross-linking. In SDS gels, the cross-linked product migrated with a slower mobility than the native protein. The cross-linked products were not detected in
the presence of dithiothreitol. Cross-linking was observed in 12 of 125 mutants. The pattern of cross-linking suggested that TM6 is close to
TMs 10, 11, and 12, while TM12 is close to TMs 4, 5, and 6. In some
mutants the presence of drug substrate colchicine, verapamil,
cyclosporin A, or vinblastine either enhanced or inhibited cross-linking. Cross-linking was inhibited in the presence of ATP plus
vanadate. These results suggest that the TM segments critical for drug
binding must be close to each other and exhibit different
conformational changes in response to binding of drug substrate or
vanadate trapping of nucleotide. Based on these results, we propose a
model for the arrangement of the TM segments.
ACCELERATED PUBLICATION
The Packing of the Transmembrane Segments of Human Multidrug
Resistance P-glycoprotein Is Revealed by Disulfide Cross-linking
Analysis*
*
This work was supported by a Grant RO1 CA80900 from the
National Institutes of Health and by grants from the Medical Research Council of Canada and the Canadian Cystic Fibrosis Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Medical Research Council Scientist and Canadian Cystic Fibrosis
Foundation Zellers' Senior Scientist. To whom correspondence should be
addressed: Dept. of Medicine, University of Toronto, Rm. 7342, Medical
Sciences Bldg., 1 King's College Circle, Toronto, Ontario M5S
1A8, Canada. Tel.:/Fax: 416-978-1105; E-mail: david.clarke@ utoronto.ca.
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