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Papers In Press, published online ahead of print December 21, 2005
Dept. of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX 77555-1031
Corresponding Author: yozhao{at}utmb.edu
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.
J. Biol. Chem, 10.1074/jbc.M511464200
Submitted on October 21, 2005
Revised on December 12, 2005
Accepted on December 21, 2005
Structure of microsomal cytochrome P450 2B4 complexed with the antifungal drug bifonazole: Insight into P450 conformational plasticity and membrane interaction
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