title: Structure of microsomal cytochrome P450 2B4 complexed with the antifungal drug bifonazole: insight into P450 conformational plasticity and membrane interaction.
abstract: 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 A 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 approximately 15 degrees 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.
authors.affiliation: Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX 77555, USA. yozhao@utmb.edu
authors.forename: Yonghong
authors.initials: Y
authors.lastname: Zhao
authors.forename: Mark A
authors.initials: MA
authors.lastname: White
authors.forename: B K
authors.initials: BK
authors.lastname: Muralidhara
authors.forename: Ling
authors.initials: L
authors.lastname: Sun
authors.forename: James R
authors.initials: JR
authors.lastname: Halpert
authors.forename: C David
authors.initials: CD
authors.lastname: Stout
mesh_terms: D000935:Antifungal Agents / Q000737:chemistry / Q000378:metabolism*; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism*; D018360:Crystallography, X-Ray; D000072467:Cytochrome P450 Family 2; D007093:Imidazoles / Q000737:chemistry / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology*; D008958:Models, Molecular; D015394:Molecular Structure; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: D000935:Antifungal Agents; D007093:Imidazoles; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit); C036596:bifonazole