The Structure of Human Microsomal Cytochrome P450 3A4 Determined by X-ray Crystallography to 2.05-Å Resolution

Abstract

The structure of P450 3A4 was determined by x-ray crystallography to 2.05-Å resolution. P450 3A4 catalyzes the metabolic clearance of a large number of clinically used drugs, and a number of adverse drug-drug interactions reflect the inhibition or induction of the enzyme. P450 3A4 exhibits a relatively large substrate-binding cavity that is consistent with its capacity to oxidize bulky substrates such as cyclosporin, statins, taxanes, and macrolide antibiotics. Family 3A P450s also exhibit unusual kinetic characteristics that suggest simultaneous occupancy by smaller substrates. Although the active site volume is similar to that of P450 2C8 (PDB code: 1PQ2), the shape of the active site cavity differs considerably due to differences in the folding and packing of portions of the protein that form the cavity. Compared with P450 2C8, the active site cavity of 3A4 is much larger near the heme iron. The lower constraints on the motions of small substrates near the site of oxygen activation may diminish the efficiency of substrate oxidation, which may, in turn, be improved by space restrictions imposed by the presence of a second substrate molecule. The structure of P450 3A4 should facilitate a better understanding of the substrate selectivity of the enzyme.Determination of the structure of P450 1 3A4 is of particular interest because the enzyme contributes extensively to human drug metabolism due to its high level of expression in liver (1) and broad capacity to oxidize structurally diverse substrates (2, 3). The enzyme also provides a significant barrier to the bioavailability of new drug candidates contributing to attrition from the developmental pipeline. Additionally, metabolic drug-drug interactions between substrates and inhibitors of the enzyme can profoundly affect the safety or efficacy of drug therapy (4, 5).Our laboratory was the first to demonstrate that microsomal P450s could be crystallized for structural determination by x-ray crystallography when the proteins were modified for expression as conditional membrane proteins (6, 7). As a result, structures for P450s in family 2, subfamilies B and C are now available (8 -14). P450s of family 3, subfamily A exhibit less than 40% amino acid sequence identity with family 2 P450s. In addition, family 3 P450s often exhibit complex kinetic properties such as substrate and effector activation. Effectors or alternative substrates can modulate the apparent binding affinity for other inhibitors (15) and substrates (16). Moreover, there are a number of examples where alternative substrates fail to inhibit the oxidation of specific substrates leading to kinetic models based on the occupancy of the substrate-binding cavity by two substrates that each can be oxidized by the reactive, hypervalent oxy-perferryl heme intermediate without interference from the other (17, 18). The observation that P450 3A4 oxidizes some of the largest substrates identified for P450s, such as cyclosporin, bromocryptine, and macrolide antibiotics (3), has generally suggested the likelihood that...

Energetics of heterotropic cooperativity between α-naphthoflavone and testosterone binding to CYP3A4

“…A conclusion made by the apparent weak temperature dependence of TST binding to CYP3A4 ( Figure 7B). This is supported by the x-ray crystal structure of CYP3A4 with [21] and without [22] progesterone, which showed that there are only very minor differences between these two structures (data not shown). Figure 9B shows the binding affect of ANF on TST binding.…”

“…The recently solved x-ray crystal structures of CYP3A4 have shed some light on the molecular basis of the first two models [20][21][22]. One structure of the enzyme showed that CYP3A4 has a large active site with a peripheral progesterone binding site, which would be consistent with the peripheral effector binding site model, although no progesterone occupied the active site [21].…”

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

“…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].…”

The Cytochrome P450 3A4 has three Major Conformations: New Clues to Drug Recognition by this Promiscuous Enzyme

“…For 4D6Z, both PK9 and the imidazole are in the active site, but only the imidazole is bonded to the heme. [11] -0 C 1W0E [12] -0 C 4I3Q [13] Water À0.65 4.2 1 C 1W0F [12] state, R212 moves toward the protein surface at the opposite of the active site, thus freeing for the ligand the space occupied in the C state by the side chain of R212 (see Figure 3). The helix I is shifted by a move of F304 and A305 away the cavity, thus creating space for the ligand.…”