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Originally published In Press as doi:10.1074/jbc.C100467200 on August 22, 2001

J. Biol. Chem., Vol. 276, Issue 40, 36877-36880, October 5, 2001
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ACCELERATED PUBLICATION
Determining the Dimensions of the Drug-binding Domain of Human P-glycoprotein Using Thiol Cross-linking Compounds as Molecular Rulers*

Tip W. Loo and David M. ClarkeDagger

From the Department of Medicine and Department of Biochemistry, Canadian Institutes for Health Research Group in Membrane Biology, University of Toronto, Toronto, Ontario M5S 1A8, Canada

The human multidrug resistance P-glycoprotein (P-gp) interacts with a broad range of compounds with diverse structures and sizes. There is considerable evidence indicating that residues in transmembrane segments 4-6 and 10-12 form the drug-binding site. We attempted to measure the size of the drug-binding site by using thiol-specific methanethiosulfonate (MTS) cross-linkers containing spacer arms of 2 to 17 atoms. The majority of these cross-linkers were also substrates of P-gp, because they stimulated ATPase activity (2.5- to 10.1-fold). 36 P-gp mutants with pairs of cysteine residues introduced into transmembrane segments 4-6 and 10-12 were analyzed after reaction with 0.2 mM MTS cross-linker at 4 °C. The cross-linked product migrated with lower mobility than native P-gp in SDS gels. 13 P-gp mutants were cross-linked by MTS cross-linkers with spacer arms of 9-25 Å. Vinblastine and cyclosporin A inhibited cross-linking. The emerging picture from these results and other studies is that the drug-binding domain is large enough to accommodate compounds of different sizes and that the drug-binding domain is "funnel" shaped, narrow at the cytoplasmic side, at least 9-25 Å in the middle, and wider still at the extracellular surface.

* This work was supported in part by National Institutes of Health Grant RO1 CA80900 and by grants from the Canadian Institutes for Health Research (CIHR) 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.

Dagger Investigator of the CIHR. To whom correspondence should be addressed: Dept. of Medicine, Rm. 7342, Medical Sciences Bldg., 1 King's College Circle, University of Toronto, Toronto, Ontario M5S 1A8, Canada. Tel./Fax: 416-978-1105.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.


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