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J. Biol. Chem., Vol. 281, Issue 9, 5973-5981, March 3, 2006
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1
2
From the
Department of Pharmacology and Toxicology and the Sealy Center for Structural Biology, University of Texas Medical Branch, Galveston, Texas 77555 and the ¶Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037
To better understand ligand-induced structural transitions in cytochrome P450 2B4, protein-ligand interactions were investigated using a bulky inhibitor. Bifonazole, a broad spectrum antifungal agent, inhibits monooxygenase activity and induces a type II binding spectrum in 2B4dH(H226Y), a modified enzyme previously crystallized in the presence of 4-(4-chlorophenyl)imidazole (CPI). Isothermal titration calorimetry and tryptophan fluorescence quenching indicate no significant burial of protein apolar surface nor altered accessibility of Trp-121 upon bifonazole binding, in contrast to recent results with CPI. A 2.3 Å crystal structure of 2B4-bifonazole reveals a novel open conformation with ligand bound in the active site, which is significantly different from either the U-shaped cleft of ligand-free 2B4 or the small active site pocket of 2B4-CPI. The O-shaped active site cleft of 2B4-bifonazole is widely open in the middle but narrow at the top. A bifonazole molecule occupies the bottom of the active site cleft, where helix I is bent 
15° to accommodate the bulky ligand. The structure also defines unanticipated interactions between helix C residues and bifonazole, suggesting an important role of helix C in azole recognition by mammalian P450s. Comparison of the ligand-free 2B4 structure, the 2B4-CPI structure, and the 2B4-bifonazole structure identifies structurally plastic regions that undergo correlated conformational changes in response to ligand binding. The most plastic regions are putative membrane-binding motifs involved in substrate access or substrate binding. The results allow us to model the membrane-associated state of P450 and provide insight into how lipophilic substrates access the buried active site.
Received for publication, October 21, 2005 , and in revised form, December 12, 2005.
The atomic coordinates and structure factors (code 2BDM) 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 part by National Institutes of Health Grants ES03619 (to J. R. H.) and GM61545 (to C. D. S.) and NIEHS Center Grant ES06676 (to J. R. H.). 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 Figs. S1 and S2.
2 Supported by Sealy and Smith Foundation for Medical Research.
1 To whom correspondence should be addressed. Tel.: 409-772-9670; Fax: 409-772-9642; E-mail: yozhao{at}utmb.edu.
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