Structure of Mammalian Cytochrome P450 2B4 Complexed with 4-(4-Chlorophenyl)imidazole at 1.9-Å Resolution

Abstract

A 1.9-Å molecular structure of the microsomal cytochrome P450 2B4 with the specific inhibitor 4-(4-chlorophenyl)imidazole (CPI) in the active site was determined by x-ray crystallography. In contrast to the previous experimentally determined 2B4 structure, this complex adopted a closed conformation similar to that observed for the mammalian 2C enzymes. The differences between the open and closed structures of 2B4 were primarily limited to the lid domain of helices F through G, helices B and C, the N terminus of helix I, and the ␤ 4 region. These large-scale conformational changes were generally due to the relocation of conserved structural elements toward each other with remarkably little remodeling at the secondary structure level. For example, the F and G helices were maintained with a sharp turn between them but are placed to form the exterior ceiling of the active site in the CPI complex. CPI was closely surrounded by residues from substrate recognition sites 1, 4, 5, and 6 to form a small, isolated hydrophobic cavity. The switch from open to closed conformation dramatically relocated helix C to a more proximal position. As a result, heme binding interactions were altered, and the putative NADPH-cytochrome P450 reductase binding site was reformed. This suggests a structural mechanism whereby ligand-induced conformational changes may coordinate catalytic activity. Comparison of the 2B4/CPI complex with the open 2B4 structure yields insights into the dynamics involved in substrate access, tight inhibitor binding, and coordination of substrate and redox partner binding. Cytochromes P450 (P450)1 are involved in steroidogenesis, fatty acid metabolism, synthesis of bile and retinoid acids, and production of plant toxins, but it is their function in the elimination of xenobiotics that has received the most attention. In mammals, xenobiotic metabolizing P450s play the central role in detoxification of hydrophobic drugs, carcinogens, and toxins by decreasing the lipid solubility of these chemicals and, thus, promoting excretion. In contrast to the strict substrate selectivity of classical enzymes, xenobiotic-metabolizing P450s can each bind and oxidize a set of substrates with distinct sizes, shapes, and stereochemical features. Although the variety of substrates binding to a given P450 is often broad, the oxidation of each is usually remarkably regiospecific and stereospecific.Identification of the structural basis for the specific monooxygenation and binding of a diverse but select set of substrates has been a particularly challenging goal, which is an important prerequisite for understanding selective substrate oxidation. Although the diversity of substrates might suggest an easily accessible active site, initial structures of both soluble bacterial (1) and microsomal mammalian (2) P450s revealed active sites buried within the globular structure of the protein.A few recent bacterial structures, however, suggest a "lid" domain composed of helices F and G, the motion of which controls substrate entry (3-5). Protein ...

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