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

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Science  30 Jul 2004:
Vol. 305, Issue 5684, pp. 683-686
DOI: 10.1126/science.1099736


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.

CYP3A4 is a complex heme-containing enzyme that exhibits non–Michaelis-Menten kinetics and both homotrophic and heterotrophic cooperativity toward several substrates (1, 2). For example, CYP3A4 exhibits atypical kinetic behavior in vitro with aflatoxin B1, amitriptyline, cabamazepine, progesterone, and diazepam (3, 4). Studies have suggested that CYP3A4 has a noncatalytic effector site within the active-site cavity, capable of modulating function. In this model, the substrate and effector molecules occupy separate positions and only the substrate molecule has access to the reactive oxygen (5). These observations, combined with the vast chemical diversity of CYP3A4 substrates, have led to speculation that CYP3A4 has an adaptive active site capable of binding multiple compounds simultaneously (610). After examining the interaction of 10 probe substrates and finding that they could be divided into groups related to their size or chemical class, Kenworthy et al. (11) proposed that three subpockets exist within the active site of CYP3A4. Additional studies have demonstrated the existence of multiple kinetically distinguishable conformations of CYP3A4, in the presence or absence of a substrate, effector, or inhibitor molecule(s) (1214), which may contribute to allostery. External factors such as the binding of cytochrome b5 and the presence of phospholipids (15) also affect the activity of CYP3A4 toward some compounds and may also stabilize particular conformations.

Understanding how this complex enzyme recognizes multiple, diverse chemical structures has been hampered by the lack of knowledge about the three-dimensional structure of CYP3A4. Homology models (16, 17) have limited use because of the substantial sequence diversity of P450s. The most widely used templates for modeling CYP3A4 are the P450 structures from Bacillus megaterium (P450 BM3) and Saccharopolyspora erythraea (P450 EryF), both of which share less than 25% sequence identity with CYP3A4. Furthermore, the sequence identity with human CYP2C9, for which the crystal structure was recently determined (18), is only 24%. We report crystal structures of CYP3A4, both unliganded and bound to either the inhibitor metyrapone or the substrate progesterone. The protein used in these studies was N-terminally truncated to facilitate crystallization and shown to be functionally active (19).

The ligand-free structure of CYP3A4 was solved to a resolution of 2.8 Å by a combination of multiple-wavelength anomalous dispersion and molecular replacement techniques (19) (tables S2 and S3). The overall structure of CYP3A4 conforms to the fold that is characteristic of the P450 superfamily, with a small, predominantly β-strand N-terminal domain and a larger, helical, C-terminal domain that contains the heme and the active site (Fig. 1A). The heme iron is ligated by a conserved cysteine (Cys442) and the propionates of the heme interact with the side chains of Arg105, Trp126, Arg130, Arg375, and Arg440. The heme is accessible to solvent by means of channels formed by β sheet 1, the B-C loop and the F-G region (Fig. 1B and fig. S1). There is a channel running from the active site to the outside of the protein formed by the B-C loop and the G′ helix, whereas another channel is formed by the F′ helix and the β sheet 1. The two channels are separated by the loop between the B′ and the C helices, a region well recognized to adopt different conformations dependent on the presence of a ligand (20). Although the heme of the protein sample used for crystallization was ferric and low spin, x-rays are known to be capable of reducing heme iron, and thus its oxidation state in the structure is unknown (21). A low-spin ferric heme iron would be expected to have a ligand or water molecule in the sixth coordination position, but the structure reveals no ordered water bound in this position. A five-coordinate heme iron would require that the iron be displaced out of the plane of the coordinating porphyrin nitrogen atoms and toward the cysteine sulfur atom. However, given the resolution of the data, no conclusions can be reliably drawn regarding coplanarity of the heme iron. Another functionally important water molecule in other P450s is located between the highly conserved residues Ala305 and Thr309, which lie on the I helix and are close to the heme. In some P450 structures, the threonine residue interacts with this water molecule, which disrupts the hydrogen-bonding pattern of the secondary structure, resulting in a kink in helix I (22). Although a similar but less pronounced kink is observed in CYP3A4, there is no discrete density visible for this water molecule.

Fig. 1.

(A) Overall fold of CYP3A4, colored from blue at the N terminus to green, to yellow, to red at the C terminus. The same coloring scheme is used for CYP3A4 in (B) and in Figs. 3 and 4 and for CYP2C9 in (C). The heme is depicted as a ball-and-stick model in the center of the molecule, flanked by helix I. Helix A′′, not present in other P450 structures, is shown at the top left of the structure, and the shortened F helix is to the right of the heme. All figures were produced with AstexViewer version 2 (36). (B) View of the Phe-cluster of CYP3A4. The solvent-accessible molecular surface of the active-surface cavity of CYP3A4 is depicted as a semitransparent green surface. The seven phenylalanine residues that make up the Phe-cluster are depicted as ball-and-stick models. The available volume of the active site is about 520 Å3 [calculated with the LIGSITE algorithm (28)]. The short F helix is shown at the right of the structure, and leads into a loop region that contains Phe213, Phe215, Phe219, and Phe220 before the F′ helix. The Phe-cluster is about 10 Å above the heme, which is depicted as a ball-and-stick model at the bottom of the structure. (C) View of the active-site cavity of CYP2C9. The solvent-accessible molecular surface of CYP2C9 (PDB entry 1OG2) is depicted as a semitransparent surface in purple. The available volume of the active site is about 600 Å3 [calculated with the LIGSITE algorithm (28)]. The F helix of CYP2C9 is longer than that of CYP3A4, and the relative position of secondary structural elements results in a narrowing of the active site toward the heme and a substantially different shape when compared with the active site of CYP3A4.

CYP3A4 has a number of features that differ from other P450 structures. A notably hydrophobic region is located around the loop following helix A′′ (top left, Fig. 1A), a helix not observed in other P450 crystal structures. This region, along with the G′ helix and the loop between the G′ and G helices, which are also hydrophobic, may mediate interaction with the microsomal membrane. Another unexpected feature of the CYP3A4 structure is the region following helix F (Fig. 1, A and B). This helix is markedly short compared with other P450 structures, and it leads into an ordered stretch of polypeptide chain that does not conform to any secondary structure motif. This region is located above and perpendicular to helix I and contains a number of residues that have been shown by site-directed mutagenesis to have a direct or indirect role in CYP3A4 function. For example, Leu210, implicated in effector binding as well as the stereo- and regioselectivity profile of CYP3A4 (7, 23, 24), lies in this region with its side chain pointing away from the active site. Leu211 and Asp214, implicated in cooperativity (23, 25), also lie in this extended loop region and point away from the active site. In contrast, the side chains of two other residues shown by mutagenesis to have no role in cooperativity (23), Phe213 and Phe215, point toward the active site and, together with another five phenylalanine residues (Phe108, Phe219, Phe220, Phe241, and Phe304), form a “Phe-cluster” (Fig. 1B). No other structurally characterized P450 has such a cluster of phenylalanine residues in this region. Some of these other Phe-cluster residues have also been shown by mutagenesis studies to be involved in CYP3A4 activity. For example, Phe304, which lies on the I helix, has a dual role in cooperativity, regioselectivity, and stereoselectivity (7, 26, 27), whereas the substitution of Phe108 with a smaller or larger amino acid affected the metabolism of some substrates (7).

The Phe-cluster lies above the active site, with the aromatic side chains stacking against each other to form a prominent hydrophobic core. This region of the structure is highly ordered with an average B factor of 41 Å2 for the residues in the cluster, compared with the average of 66 Å2 over the entire structure. Furthermore, as a result of this aromatic clustering, the active site of CYP3A4 has a probe-accessible volume (28) of about 520 Å3, substantially smaller than expected on the basis of its reported metabolism of large substrates. The overall volume of the CYP3A4 active site is also smaller than that of CYP2C9 (18), which is 670 Å3, and different in shape (Fig. 1, B and C). The variation in the active-site topologies is a consequence of the Phe-cluster, which is positioned on the top of the active site closer to the heme, with β sheet 1 farther from the heme compared with CYP2C9 (Fig. 1C). An overlay of CYP3A4 with the structures of P450 BM3 (29) and P450 EryF (30) (Fig. 2) shows the limitations of homology models based on these two bacterial structures. The position of the Phe-cluster results in a closed conformation of the active site of CYP3A4 that is similar to the closed conformation adopted by P450 EryF (30), but the spatial arrangement of the F-G loop and F helices is not conserved (Fig. 2). The heme of CYP3A4 has greater accessibility to the active site than does that of CYP2C9 (Fig. 1C), which could allow two substrate molecules to have access to the reactive oxygen, consistent with data indicating that CYP3A4 is able to bind and metabolize multiple substrate molecules simultaneously (6, 8). Conformational movement involving the Phe-cluster could also reposition phenylalanine residues, perhaps resulting in an extension of helix F and a larger active site. There is a precedent for such a mechanism from the CYP119 structure in which, although there is no analogous Phe-cluster, ligand binding resulted in an extension of helix F and movement of the G helix and the intervening F-G loop (31). To investigate whether conformational movement is a necessary prerequisite for ligand binding by CYP3A4, we determined the crystal structure in complex with the known inhibitor metyrapone. The metyrapone cocomplex structure was determined with the use of both soaking and, more importantly, cocrystallization techniques, which allowed conformational movement by the protein if required.

Fig. 2.

Stereoimage showing the structural overlay of CYP3A4 in red, with P450 BM3 (molecule A of PDB entry 1BU7) in blue, and P450 EryF (PDB entry 1OXA) in yellow. The heme is depicted as a ball-and-stick model toward the bottom of the figure. The F helix of CYP3A4 (red) is notably shorter than the F helices of P450 BM3 (blue) or P450 EryF (yellow). CYP3A4 has two helices F′ and G′ that are absent in P450 BM3, P450 EryF, and other bacterial P450 structures.

Contrary to expectations, the binding of metyrapone to CYP3A4 reveals essentially no conformational changes in the protein. Ultraviolet and visible spectroscopic data (32) indicate that in solution the inhibitor ligates the heme. This is consistent with the binding mode observed in the crystal structure, in which metyrapone is bound directly to the heme iron by means of the alkylpyridine nitrogen (Fig. 3), and exhibits good shape complementarity with the CYP3A4 active site (fig. S2). An alternative binding mode for metyrapone may involve the nitrogen atom of the keto-pyridine group, as observed previously in the cocomplex with bacterial P450 cam (33). However, the electron density for this CYP3A4 cocomplex indicates that the illustrated binding mode is predominant. The volume occupied by the metyrapone molecule is 230 Å3, leaving sufficient space for additional molecules to bind within the active site.

Fig. 3.

Stereoview of metyrapone bound to the heme of CYP3A4. The metyrapone, heme, and surrounding residues are depicted as ball-and-stick models. The initial sigmaA-weighted Fo-Fc electron density map is shown in red contoured at the 2.0σ level, and the final sigma-weighted Fo-Fc electron density map, calculated omitting the heme, is shown in red contoured at the 2.0σ level, and then is shown in blue contoured at the 1.0σ level. The compound exhibits a single binding orientation and interacts with the protein by means of a pyridinyl group of metyrapone.

Although formation of the metyrapone complex is achieved with little conformational change, we anticipate that substantial protein movement, possibly involving the F and G helical regions and the Phe-cluster, would be required to permit the entry and accommodation of larger compounds. Movement of the B-C loop region could also open up the channel and increase the volume of the active site. To investigate this possibility and explore how CYP3A4 recognizes substrates, we determined the structure of the complex with progesterone by cocrystallization. Surprisingly, progesterone also induced very little conformational change in the protein and bound at a peripheral site very close to the Phe-cluster (Fig. 4) and some distance away (>17 Å) from the heme iron. The initial electron density maps indicated that one progesterone molecule bound in a single clearly defined orientation (fig. S3), as reflected by an average B factor for progesterone of 45 Å2, compared with 61 Å2 for the protein. The progesterone molecule interacts with the protein through a hydrogen bond between its acetyl oxygen and the amide nitrogen of Asp214, packs against the side chains of Phe219 and Phe220 in the Phe-cluster (fig. S3), and appears fairly accessible to solvent. Although this binding site may be an artifact of crystallization, we argue that it is likely to have functional relevance. This is supported by the observation that the side chains of Leu211, Phe213, and Asp214 residues implicated in both progesterone homotrophic and heterotrophic cooperativity (5, 25) lie in the vicinity of this peripheral binding pocket. On the basis of these findings, we propose that the progesterone-binding site may be involved in the recognition of effector as well as substrate molecules and has a role in modulating cooperativity. Notably, although progesterone can be docked into the active site of CYP3A4 in an orientation consistent with its metabolism, there is no evidence of progesterone binding within the active-site cavity in this structure.

Fig. 4.

Peripheral binding of progesterone to CYP3A4. The solvent-accessible surface of the progesterone molecule is shown in yellow at the top of the figure. A cross section of the solvent-accessible surface of CYP3A4 is shown in blue, revealing the active-site cavity of CYP3A4 in the center of the figure. Progesterone sits in a shallow pocket on top of the Phe-cluster of CYP3A4, separated from the heme by the F-G region of the molecule. Progesterone binds 17 Å above the heme iron and on the face of CYP3A4 most likely to interact with the membrane.

Several studies indicate that the F-G and B-C loops of P450, which constitute the putative substrate-access channel, could contribute to membrane binding and orient the substrate-access channel toward the membrane surface (34, 35). The location of the progesterone-binding site in the F-G region is consistent with a role in initial substrate recognition, because it lies along an appropriate route for compounds that move directly from the membrane into the active site of CYP3A4. Appropriate conformational movement of residues around the Phe-cluster could result in a substrate-access channel that stretched from this peripheral binding site to the heme group, providing a route for a compound to move from this initial recognition site to the active site. Such conformational movements in the Phe-cluster could be triggered by interaction with a physiological electron transfer partner such as cytochrome b5 or cytochrome P450 reductase, or by a change in membrane properties to allow the substrate molecule to move from its surface position to a position in the active site that is suitable for metabolism. Future studies that investigate these potential mechanisms of molecular recognition and enzyme activity can now be guided by the crystal structure of CYP3A4.

Supporting Online Material

Materials and Methods

Figs. S1 to S3

Tables S1 to S3


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