Structural features of cytochromes P450 and ligands that affect drug metabolism as revealed by X-ray crystallography and NMR

Abstract

Cytochromes P450 (P450s) play a major role in the clearance of drugs, toxins, and environmental pollutants. Additionally, metabolism by P450s can result in toxic or carcinogenic products. The metabolism of pharmaceuticals by P450s is a major concern during the design of new drug candidates. Determining the interactions between P450s and compounds of very diverse structures is complicated by the variability in P450-ligand interactions. Understanding the protein structural elements and the chemical attributes of ligands that dictate their orientation in the P450 active site will aid in the development of effective and safe therapeutic agents. The goal of this review is to describe P450-ligand interactions from two perspectives. The first is the various structural elements that microsomal P450s have at their disposal to assume the different conformations observed in X-ray crystal structures. The second is P450-ligand dynamics analyzed by NMR relaxation studies.

Keywords: Cytochrome P450; NMR; P450; X-ray crystallography; drug metabolism; mammalian; microsomal; plasticity; protein structure; xenobiotic.

Figure 1

A ribbon diagram of the CYP2B6–4-CPI complex (PDB ID 3IBD) shows the typical tertiary structure of a cytochrome P450. The twelve α-helices (A–L) and four β-sheets (1–4) are labeled, with minor helices denoted as 'prime' or 'double prime'. The protein is colored from blue (N-terminus) to red (C-terminus), while the heme and 4-CPI inhibitor are shown as red and cyan sticks, respectively.

Figure. 2

A C_α overlay of the methoxsalen (red, PDB ID 1Z11), coumarin (light green, PDB ID 1Z10), and 4, 4'-dipyridyl disulfide (dark blue, PDB ID 2FDY) complexes of CYP2A6. The enzyme requires very little structural rearrangement to bind these ligands. Gray mesh shows the active site cavity of the CYP2A6–coumarin complex and is representative of the cavity of other CYP2A6 structures. Stick diagrams (bottom) show the chemical structures of the ligands bound to each enzyme complex.

Figure 3

Ribbon diagrams of the four markedly different conformations observed in CYP2B4 X-ray crystal structures. CYP2B4 forms a closed, compact structure when bound to the small inhibitor (A) 4-CPI (PDB ID 1SUO). The B', C, F, F', G', and H helices and the N-terminus of the I helix move to accomodate the larger inhibitors (B) 1-PBI (PDB ID 3G5N) and (C) bifonazole (PDB ID 2BDM) and (D) in the absence of ligand (PDB ID 1PO5). Stick diagrams show the chemical structures of inhibitors.

Figure 4

A comparison of the active site cavities of the 4-CPI complexes of CYP2B6 (red, PDB ID 3IBD) and 2B4 (green, PDB ID 1SUO). Despite their structural similarities (RMSD 0.65 Å), the two enzymes differ with respect to their active site cavity volumes (red and green mesh). The larger cavity in CYP2B6 is created by the movement of E301 out of the active site, the rotations of I101 and V477, and the the change from phenylalanine to methionine at position 365.

Figure 5

The T- or Y-shaped active site cavity (gray mesh) of the CYP2C8–montelukast complex (PDB ID 2NNI) is shown with three branches of varying lengths. The cavity is able to accommodate CYP2C8's various substrates by allowing them to bind in one or more of these branches. Montelukast (green sticks) fully occupies branches 2 and 3 and a portion of branch 1.

Figure 6

C_α overlay of ligand free CYP3A4 (blue, PDB ID 1TQN) and the CYP3A4–ketoconazole complex (yellow, PDB ID 2V0M). This ligand free structure is similar to a ligand free structure (PDB ID 1W0E) from another study and the CYP3A4 complexes of progesterone (PDB ID 1W0F) and metyrapone (PDB ID 1W0G). In this overlay, static portions of the enzyme (gray) do not change, but the B', F, F', G', and H helices and the N-terminus of the I helix alter their structures in response to the binding of two ketoconazole molecules. In particular, the F/F' loop changes position by ~5.5 Å.