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J. Biol. Chem., Vol. 283, Issue 25, 17227-17237, June 20, 2008

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Determinants of Cytochrome P450 2C8 Substrate Binding

STRUCTURES OF COMPLEXES WITH MONTELUKAST, TROGLITAZONE, FELODIPINE, AND 9-CIS-RETINOIC ACID^*

Guillaume A. Schoch¹, Jason K. Yano², Stefaan Sansen, Patrick M. Dansette, C. David Stout^¶, and Eric F. Johnson³

From the Departments of Molecular and Experimental Medicine and ^¶Molecular Biology, The Scripps Research Institute, La Jolla, California 92037 and the Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, CNRS UMR 8601, Université Paris Descartes, 45 Rue des Saints Pères, 75270 Paris Cedex 06, France

Although a crystal structure and a pharmacophore model are available for cytochrome P450 2C8, the role of protein flexibility and specific ligand-protein interactions that govern substrate binding are poorly understood. X-ray crystal structures of P450 2C8 complexed with montelukast (2.8 Å), troglitazone (2.7 Å), felodipine (2.3 Å), and 9-cis-retinoic acid (2.6 Å) were determined to examine ligand-protein interactions for these chemically diverse compounds. Montelukast is a relatively large anionic inhibitor that exhibits a tripartite structure and complements the size and shape of the active-site cavity. The inhibitor troglitazone occupies the upper portion of the active-site cavity, leaving a substantial part of the cavity unoccupied. The smaller neutral felodipine molecule is sequestered with its dichlorophenyl group positioned close to the heme iron, and water molecules fill the distal portion of the cavity. The structure of the 9-cis-retinoic acid complex reveals that two substrate molecules bind simultaneously in the active site of P450 2C8. A second molecule of 9-cis-retinoic acid is located above the proximal molecule and can restrain the position of the latter for more efficient oxygenation. Solution binding studies do not discriminate between cooperative and noncooperative models for multiple substrate binding. The complexes with structurally distinct ligands further demonstrate the conformational adaptability of active site-constituting residues, especially Arg-241, that can reorient in the active-site cavity to stabilize a negatively charged functional group and define two spatially distinct binding sites for anionic moieties of substrates.

Received for publication, March 19, 2008 , and in revised form, April 14, 2008.

The atomic coordinates and structure factors (codes 2nnh, 2nni, 2nnj, and 2vn0) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported, in whole or in part, by National Institutes of Health Grant GM031001 (to E. F. J.). This work was also supported by Pfizer Global Research and Development and the Sam and Rose Stein Charitable Trust. Portions of this work were carried out at the Stanford Synchrotron Radiation Laboratory, a national user facility operated by Stanford University on behalf of the United States Department of Energy, Office of Basic Energy Sciences. The Stanford Synchrotron Radiation Laboratory Structural Molecular Biology Program is supported by the United States Department of Energy, Office of Biological and Environmental Research, by the National Center for Research Resources Biomedical Technology Program, and by NIGMS, National Institutes of Health. 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 material.

This article was selected as a Paper of the Week.

¹ Present address: F. Hoffmann-La Roche Ltd., Pharma Discovery Research Basel, CH-4070 Basel, Switzerland.

² Present address: Dept. of Structural Biology, Takeda San Diego, San Diego, CA 92121.

³ To whom correspondence should be addressed: Dept. of Molecular and Experimental Medicine, The Scripps Research Inst., 10550 N. Torrey Pines Rd., MEM-255, La Jolla, CA 92037. Tel.: 858-784-7918; Fax: 858-784-7978; E-mail: johnson{at}scripps.edu.

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