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J. Biol. Chem., Vol. 279, Issue 9, 7692-7697, February 27, 2004

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Disulfide Cross-linking Analysis Shows That Transmembrane Segments 5 and 8 of Human P-glycoprotein Are Close Together on the Cytoplasmic Side of the Membrane^*

Tip W. Loo, M. Claire Bartlett, and David M. Clarke

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

Human P-glycoprotein (P-gp) transports a wide variety of structurally diverse compounds out of the cell. Knowledge about the packing of the transmembrane (TM) segments is essential for understanding the mechanism of drug recognition and transport. We used cysteine-scanning mutagenesis and disulfide cross-linking analysis to determine which TM segment in the COOH half of P-gp was close to TMs 5 and 6 since these segments in the NH₂ half are important for drug binding. An active Cys-less P-gp mutant cDNA was used to generate 240 double cysteine mutants that contained 1 cysteine in TMs 5 or 6 and another in TMs 7 or 8. The mutants were subjected to oxidative cross-linking analysis. No disulfide cross-linking was observed in the 140 TM6/TM7 or TM6/TM8 mutants. By contrast, cross-linking was detected in several P-gp TM5/TM8 mutants. At 4 °C, when thermal motion is low, P-gp mutants N296C(TM5)/G774C(TM8), I299C(TM5)/F770C(TM8), I299C(TM5)/G774C(TM8), and G300C(TM5)/F770C(TM8) showed extensive cross-linking with oxidant. These mutants retained drug-stimulated ATPase activity, but their activities were inhibited after treatment with oxidant. Similarly, disulfide cross-linking was inhibited by vanadate trapping of nucleotide. These results indicate that significant conformational changes must occur between TMs 5 and 8 during ATP hydrolysis. We revised the rotational symmetry model for TM packing based on our results and by comparison to the crystal structure of MsbA (Chang, G. (2003) J. Mol. Biol. 330, 419–430) such that TM5 is adjacent to TM8, TM2 is adjacent to TM11, and TMs 1 and 7 are next to TMs 6 and 12, respectively.

Received for publication, October 28, 2003 , and in revised form, December 10, 2003.

^* This work was supported in part by National Institutes of Health Grant CA80900 and by grants from the Canadian Institutes for Health Research). 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 the Canada Research Chair in Membrane Biology. 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. or Fax: 416-978-1105; E-mail: david.clarke{at}utoronto.ca.

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