Crystal structure of a cytochrome P450 2B6 genetic variant in complex with the inhibitor 4-(4-chlorophenyl)imidazole at 2.0-A resolution

Abstract

The structure of the K262R genetic variant of human cytochrome P450 2B6 in complex with the inhibitor 4-(4-chlorophenyl)imidazole (4-CPI) has been determined using X-ray crystallography to 2.0-A resolution. Production of diffraction quality crystals was enabled through a combination of protein engineering, chaperone coexpression, modifications to the purification protocol, and the use of unique facial amphiphiles during crystallization. The 2B6-4-CPI complex is virtually identical to the rabbit 2B4 structure bound to the same inhibitor with respect to the arrangement of secondary structural elements and the placement of active site residues. The structure supports prior P450 2B6 homology models based on other mammalian cytochromes P450 and is consistent with the limited site-directed mutagenesis studies on 2B6 and extensive studies on P450 2B4 and 2B1. Although the K262R genetic variant shows unaltered binding of 4-CPI, altered binding affinity, kinetics, and/or product profiles have been previously shown with several other ligands. On the basis of new P450 2B6 crystal structure and previous 2B4 structures, substitutions at residue 262 affect a hydrogen-bonding network connecting the G and H helices, where subtle differences could be transduced to the active site. Docking experiments indicate that the closed protein conformation allows smaller ligands such as ticlopidine to bind to the 2B6 active site in the expected orientation. However, it is unknown whether 2B6 undergoes structural reorganization to accommodate bulkier molecules, as previously inferred from multiple P450 2B4 crystal structures.

Fig. 1.

Divergent stereo view of the 2B6-4-CPI complex. The sequence is colored from the N terminus (blue) to the C terminus (red). The heme and partial Cymal-5 detergents are shown as red sticks. The 4-CPI inhibitor is shown as cyan sticks.

Fig. 2.

Protein sequence alignment of 2B4dH and 2B6dH(Y226H, K262R). The alignment also includes the N terminus of wild-type 2B6 to indicate changes made to the construct for expression and purification. Secondary structure of the 4-CPI complexes is indicated by boxes (α-helices) and underlined regions (β-strands). Mutations are marked in bold type. Diamonds indicate residues interacting with 4-CPI. The locations of known 2B6 SNPs are denoted by squares.

Fig. 3.

Divergent stereo view of an overlay of both 2B4 (purple) and 2B6 (green) 4-CPI complexes. The 2B6 heme and 4-CPI are shown in red, whereas the corresponding molecules in 2B4 are shown in blue. a, the overall structure of the human and rabbit enzymes, with an RMSD of 0.65 Å, are almost indistinguishable from one another. b, the active sites of 2B4 and 2B6 are shown. Residues found within a 5-Å radius of 4-CPI are depicted as sticks. An F_o − F_c simulated annealing omit map contoured at 3-σ clearly shows the 2B6 4-CPI and heme.

Fig. 4.

Ribbon-and-stick depiction of a typical pose for ticlopidine docking into the active site of a model of P450 2B6 based on the crystal structure of the 4-CPI complex. This and other similar poses are consistent with data that show ticlopidine to be oxidized on the thiophene ring and cause mechanism-based inactivation of P450 2B6.

Fig. 5.

Ribbon diagram showing the location of known SNPs (yellow sticks) in 2B6. Residues 99 and 476 (both glycine) are not shown as sticks, but locations are identified. The heme is shown as red sticks; 4-CPI, cyan sticks. The majority of the known 2B6 coding sequence variants contain substitutions that occur relatively far from the active site, and none of them actually lies within the active site.

Fig. 6.

Ribbon-and-stick diagram depicting the G and H helices region in the 2B4 bifonazole (orange; 2bdm), 1-(4-phenyl)benzylimidazole (1-PBI) (blue; 3g5n), and 4-CPI (green; 1suo) structures. Aligning the G and H helices in the absence of the remaining protein shows that this region maintains a consistent local structure despite larger movements in the overall protein (inset). The hydrogen-bonding network among His 252, Thr 255, Arg 262, Asp 263, and Asp 266 seems to link the G and H helices together throughout these shifts in secondary structure placement. These same interactions are also seen in the present 2B6 structure.