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J. Biol. Chem., Vol. 283, Issue 9, 5769-5779, February 29, 2008

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Nucleotide-induced Structural Changes in P-glycoprotein Observed by Electron Microscopy^*

Jyh-Yeuan Lee¹, Ina L. Urbatsch, Alan E. Senior^¶, and Stephan Wilkens^||2

From the Department of Biochemistry, University of California, Riverside, California 92521, the Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, the ^¶Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642, and the ^||Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York 13210

P-glycoprotein (Pgp) is an ATP hydrolysis driven multidrug efflux pump, which, when overexpressed in the plasma membrane of certain cancers, can lead to the failure of chemotherapy. Previously, we have presented a projection structure of nucleotide-free mouse Pgp from electron microscopic images of lipid monolayer-generated two-dimensional crystals ( Lee, J. Y., Urbatsch, I. L., Senior, A. E., and Wilkens, S. (2002) J. Biol. Chem. 277, 40125-40131[Abstract/Free Full Text] ). Here we have analyzed the structure of cysteine-free human Pgp from two-dimensional crystals that were generated with the same lipid-monolayer technique in the absence and presence of various nucleotides. The images show that human Pgp has a similar structure to the mouse protein. Furthermore, the analysis of projection structures obtained under different nucleotide conditions suggests that Pgp can exist in at least two major conformations, one of which shows a central cavity between the N- and C-terminal halves of the molecule and another in which the two halves have moved sideways, thereby closing the central cavity. Intermediate conformations were observed for some nucleotide/vanadate combinations. A low-resolution, three-dimensional model of human Pgp was calculated from tilted specimen crystallized in the presence of the non-hydrolyzable nucleotide analog, adenosine 5'-O-(thiotriphosphate). The structural analysis presented here adds to the emerging picture that multidrug ABC transporters function by switching between two major conformations in a nucleotide-dependent manner.

Received for publication, August 22, 2007 , and in revised form, November 26, 2007.

^* This work was supported by National Institutes of Health Grants CA100246 (to S. W.) and GM50156 (to A. E. S.) and the Wilson Foundation, Dallas (to I. L. U.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2 and Figs. S1-S5.

¹ This work is based on a dissertation (J.-Y. L.) submitted to fulfill in part the requirement for the degree of Doctor of Philosophy at the University of California, Riverside (registration number TX-6-388-452 at the United States Copyright Office). Present address: Texas Tech University Health Sciences Center, Cell Biology and Biochemistry, Lubbock, TX 79430.

² To whom correspondence should be addressed: 750 East Adams St., Syracuse, NY 13210. Tel.: 315-464-8703; Fax: 315-464-8750; E-mail: wilkenss{at}upstate.edu.

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