title: Ketoconazole-induced conformational changes in the active site of cytochrome P450eryF.
abstract: The azole-based P450 inhibitor ketoconazole is used to treat fungal infections and functions by blocking ergosterol biosynthesis in yeast. Ketoconazole binds to mammalian P450 enzymes and this can result in drug-drug interactions and lead to liver damage. To identify protein-drug interactions that contribute to binding specificity and affinity, we determined the crystal structure of ketoconazole complexed with P450eryF. In the P450eryF/ketoconazole structure, the azole moiety and nearby rings of ketoconzole are positioned in the active site similar to the substrate, 6-deoxyerythronolide B, with the azole nitrogen atom coordinated to the heme iron atom. The remainder of the ketoconazole molecule extends into the active-site pocket, which is occupied by water in the substrate complex. Binding of ketoconazole led to unexpected conformational changes in the I-helix. The I-helix cleft near the active site has collapsed with a helical pitch of 5.4 A compared to 6.6 A in the substrate complex. P450eryF/ketoconazole crystals soaked in 6-deoxyerythronolide B to exchange ligands exhibit a structure identical with that of the original P450eryF/substrate complex, with the I-helix cleft restored to a pitch of 6.6 A. These findings indicate that the I-helix region of P450eryF is flexible and can adopt multiple conformations. An improved understanding of the flexibility of the active-site region of cytochrome P450 enzymes is important to gain insight into determinants of ligand binding/specificity as well as to evaluate models for catalytic mechanism based on static crystal structures.
authors.affiliation: Department of Chemistry and Biochemistry, California State University, Fullerton, 800 N. State College Blvd., Fullerton, CA 92834, USA. jvickery@fullerton.edu
authors.forename: J R
authors.initials: JR
authors.lastname: Cupp-Vickery
authors.forename: C
authors.initials: C
authors.lastname: Garcia
authors.forename: A
authors.initials: A
authors.lastname: Hofacre
authors.forename: K
authors.initials: K
authors.lastname: McGee-Estrada
mesh_terms: D000818:Animals; D000935:Antifungal Agents / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D001426:Bacterial Proteins; D001665:Binding Sites / Q000187:drug effects; D002384:Catalysis; D018360:Crystallography, X-Ray; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism*; D004347:Drug Interactions; D007654:Ketoconazole / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D008024:Ligands; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D008956:Models, Chemical; D008958:Models, Molecular; D015394:Molecular Structure; D018583:Pliability / Q000187:drug effects; D011485:Protein Binding; D017433:Protein Structure, Secondary / Q000187:drug effects; D013379:Substrate Specificity
chemical_list: D000935:Antifungal Agents; D001426:Bacterial Proteins; D065607:Cytochrome P-450 Enzyme Inhibitors; D008024:Ligands; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C054444:eryF protein, Saccharopolyspora erythraea; D007654:Ketoconazole