Crystal Structures of Human Cytochrome P450 3A4 Bound to Metyrapone and Progesterone

Abstract

Cytochromes P450 (P450s) metabolize a wide range of endogenous compounds and xenobiotics, such as pollutants, environmental compounds, and drug molecules. The microsomal, membrane-associated, P450 isoforms CYP3A4, CYP2D6, CYP2C9, CYP2C19, CYP2E1, and CYP1A2 are responsible for the oxidative metabolism of more than 90% of marketed drugs. Cytochrome P450 3A4 (CYP3A4) metabolizes more drug molecules than all other isoforms combined. Here we report three crystal structures of CYP3A4: unliganded, bound to the inhibitor metyrapone, and bound to the substrate progesterone. The structures revealed a surprisingly small active site, with little conformational change associated with the binding of either compound. An unexpected peripheral binding site is identified, located above a phenylalanine cluster, which may be involved in the initial recognition of substrates or allosteric effectors.

Role of subunit interactions in P450 oligomers in the loss of homotropic cooperativity in the cytochrome P450 3A4 mutant L211F/D214E/F304W

“…Recent work by Isin and Guengerich proposed a three-step binding model in which the first step does not perturb the heme spectrum and involves binding at a site peripheral to the active site [44]. This hypothesis is consistent with the observation of a peripheral progesterone binding site in a CYP3A4 X-ray crystal structure [54].…”

“…In the first structure, progesterone binds in the vicinity of L211 and D214 [54]. Here, similar to the substrate-free enzyme [54,66], the side chains of these residues point away from the active site. L211 and D214 are close to a Phe cluster containing Phe-213, Phe-215, Phe-219, Phe-220, Phe-241, and Phe-304, which is suggested to comprise a flexible region and a dimer interface [68].…”

“…These results suggested a complex relationship between substrate and effector binding, oxidation, and action. However, further progress was not possible until the very recent development of advanced spectroscopic methods for investigating multiple ligand binding to CYP3A4 [19,48] and the elucidation of ligand free and ligand bound x-ray crystal structures [11,54,66].…”

“…For this purpose, we examined an x-ray crystal structure of CYP3A4 with progesterone bound at a peripheral binding site [54] and a more recent structure with two ketoconazole molecules bound in the active site [11]. In the first structure, progesterone binds in the vicinity of L211 and D214 [54]. Here, similar to the substrate-free enzyme [54,66], the side chains of these residues point away from the active site.…”

“…In the present study, we applied our recent spectroscopic approaches to the CYP3A4 mutant L211F/D214E/F304W (FEW), which displays no homotropic cooperativity of progesterone hydroxylation and abolished heterotropic activation by ANF [55]. Interestingly, two of the residues that are altered in this mutant, L211 and D214, reside in the proposed peripheral progesterone binding site [54], whereas in the ketoconazole-bound structure, L211 and F304 contact the proximal ligand molecule [11]. To gain insight into the basis for lost cooperativity in CYP3A4 FEW, we studied the interactions of the enzyme with 1-PB, bromocriptine (BCT), and ANF monitored by absorbance spectroscopy.…”