Structural basis for ligand promiscuity in cytochrome P450 3A4

Abstract

Cytochrome P450 (CYP) 3A4 is the most promiscuous of the human CYP enzymes and contributes to the metabolism of Ϸ50% of marketed drugs. It is also the isoform most often involved in unwanted drug-drug interactions. A better understanding of the molecular mechanisms governing CYP3A4 -ligand interaction therefore would be of great importance to any drug discovery effort. Here, we present crystal structures of human CYP3A4 in complex with two well characterized drugs: ketoconazole and erythromycin. In contrast to previous reports, the protein undergoes dramatic conformational changes upon ligand binding with an increase in the active site volume by >80%. The structures represent two distinct open conformations of CYP3A4 because ketoconazole and erythromycin induce different types of coordinate shifts. The binding of two molecules of ketoconazole to the CYP3A4 active site and the clear indication of multiple binding modes for erythromycin has implications for the interpretation of the atypical kinetic data often displayed by CYP3A4. The extreme flexibility revealed by the present structures also challenges any attempt to apply computational design tools without the support of relevant experimental data.drug metabolism ͉ structural flexibility ͉ x-ray crystallography ͉ inhibitor ͉ substrate C ytochrome P450 3A4 (CYP3A4) is the most abundant of the xenobiotic-metabolizing CYP isoforms, and interactions with CYP3A4 must always be taken into consideration during the development of new medications (1).In recent years, a number of structures of mammalian CYP isoforms, including CYP2C5 (2), CYP2C9 (3, 4), CYP2C8 (5), CYP2B4 (6), CYP2A6 (7), and CYP3A4 (8, 9) have been solved. All of these structures were determined from modified versions of the protein where the N-terminal transmembrane helix was truncated and, in some cases, a number of mutations aimed at increasing solubility were introduced. The mammalian CYP structures all adopt the general CYP fold first described in 1987 when the structure of the bacterial P450 CYP101 was determined by x-ray crystallography (10).The ligand-free structure of CYP3A4 was published in 2004 by two independent groups (8, 9). These structures are very similar (11). The most remarkable features are the short F and G helices (nomenclature adapted from Poulos et al.; ref. 10) and a large, highly ordered hydrophobic core of phenyl alanine residues above the active site (8, 9). CYP3A4 is known to metabolize large substrates such as bromocriptine (M r 655 Da) and cyclosporine (M r 1,203 Da). A number of studies also indicate that CYP3A4 displays ligand binding that does not follow Michaelis-Menten type kinetics, and it has been suggested that two or more ligand molecules can bind to the CYP3A4 active site simultaneously (12-15). In light of these observations, the volume of the active site in the published ligand-free structures is smaller than expected, comparable with the active-site cavity seen in CYP2C9 and CYP2C8, which led Williams et al. (8) to speculate that conformational changes may o...

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

“…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]. Another structure shows two ketoconazole drug molecules bound in the active site of CYP3A4 simultaneously, which would be consistent with the multi-substrate binding model and was observed previously in the CYP3A4 homolog, cytochrome P450 eryF (P450 eryF ) [20,23]. Neither structure provides conclusive evidence for the mechanism of cooperativity, since either the substrate was absent from the active site, the ligand was inhibitory, or the concentrations used can not be extrapolated to the solution experiments.…”

“…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].…”

“…CYP3A4 has been modeled with two [11,12,14,15,[24][25][26], three [16,[27][28][29], and more ligand [13] binding sites. Since the x-ray crystal structures of CYP3A4 and P450 eryF have at most 2 ligands bound simultaneously [20,23], the data in this study were modeled with two ligand binding sites.…”

Selective, competitive and mechanism-based inhibitors of human cytochrome P450 2J2

“…In the human genome, 57 genes have been found to code for CYPs [2]. The main CYPs implicated in drug metabolism, such as CYP3A4, CYP2C9 or CYP2D6, and those responsible for the biosynthesis of steroid hormones have been extensively studied, and several x-ray structures of human CYPs have been recently published [3][4][5][6][7][8][9][10]. Much less is known about more recently discovered human CYPs such as CYP2J2 [2,11,12].…”

A Quantitative Index of Substrate Promiscuity

“…However, many enzymes are catalytically promiscuous, and they can metabolize structurally distinct substrates or convert a single substrate to multiple products. It is increasingly well-appreciated that functional promiscuity (4) is important for the evolution of new protein functions (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15), the in vitro engineering of biocatalysts (16)(17)(18)(19), and drug metabolism (8,(20)(21)(22)(23)(24)(25).…”