Cupp-Vickery J R, Garcia C, Hofacre A, McGee-Estrada K
Department of Chemistry and Biochemistry, California State University, Fullerton, 800 N. State College Blvd., Fullerton, CA 92834, USA.
J Mol Biol. 2001 Aug 3;311(1):101-10. doi: 10.1006/jmbi.2001.4803.
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.
基于唑类的细胞色素P450抑制剂酮康唑用于治疗真菌感染，其作用机制是阻断酵母中的麦角固醇生物合成。酮康唑与哺乳动物的细胞色素P450酶结合，这可能导致药物相互作用并引起肝损伤。为了确定有助于结合特异性和亲和力的蛋白质-药物相互作用，我们测定了酮康唑与P450eryF复合的晶体结构。在P450eryF/酮康唑结构中，酮康唑的唑基部分和附近的环位于活性位点，类似于底物6-脱氧红霉内酯B，唑氮原子与血红素铁原子配位。酮康唑分子的其余部分延伸到活性位点口袋中，该口袋在底物复合物中被水占据。酮康唑的结合导致I-螺旋发生意外的构象变化。活性位点附近的I-螺旋裂隙已经塌陷，螺旋间距为5.4 Å，而底物复合物中的螺旋间距为6.6 Å。浸泡在6-脱氧红霉内酯B中以交换配体的P450eryF/酮康唑晶体显示出与原始P450eryF/底物复合物相同的结构，I-螺旋裂隙恢复到6.6 Å的间距。这些发现表明P450eryF的I-螺旋区域是灵活的，可以采用多种构象。更好地理解细胞色素P450酶活性位点区域的灵活性对于深入了解配体结合/特异性的决定因素以及评估基于静态晶体结构的催化机制模型非常重要。
