Crystal structures of substrate-bound and substrate-free cytochrome P450 46A1, the principal cholesterol hydroxylase in the brain

Abstract

By converting cholesterol to 24S-hydroxycholesterol, cytochrome P450 46A1 (CYP46A1) initiates the major pathway for cholesterol removal from the brain. Two crystal structures of CYP46A1 were determined. First is the 1.9-A structure of CYP46A1 complexed with a high-affinity substrate cholesterol 3-sulfate (CH-3S). The second structure is that of the substrate-free CYP46A1 at 2.4-A resolution. CH-3S is bound in the productive orientation and occupies the entire length of the banana-shaped hydrophobic active-site cavity. A unique helix B'-C loop insertion (residues 116-120) contributes to positioning cholesterol for oxygenation catalyzed by CYP46A1. A comparison with the substrate-free structure reveals substantial substrate-induced conformational changes in CYP46A1 and suggests that structurally distinct compounds could bind in the enzyme active site. In vitro assays were performed to characterize the effect of different therapeutic agents on cholesterol hydroxylase activity of purified full-length recombinant CYP46A1, and several strong inhibitors and modest coactivators of CYP46A1 were identified. Structural and biochemical data provide evidence that CYP46A1 activity could be altered by exposure to some therapeutic drugs and potentially other xenobiotics.

Fig. 1.

CYP46A1 active site. (a) The composite-omit 2|F_o|−|F_c| electron density map (green mesh) contoured at 1.5σ around the heme (in pink) and CH-3S (in yellow). Amino acid residues (in cyan) within 4 Å of CH-3S are shown. The heme iron and water molecule 732 are represented as big brown and small red spheres, respectively. The oxygen, nitrogen, and sulfur atoms are in red, blue, and orange, respectively. Dashed cyan lines indicate hydrogen bonds. Residues forming a circular scaffold are labeled in red. (b) Enlarged view of the active site around the sulfate anion of CH-3S and (c) in the vicinity of the heme iron. Dashed gray lines connect the C24 and C25 of CH-3S and the heme iron.

Fig. 2.

Superposition of CH-3S-bound CYP46A1 structure (colored from blue at the N terminus to red at the C terminus) and vitamin D₃-bound CYP2R1 structure (in wheat) shown in stereoview. CH-3S and vitamin D₃ are in yellow and cyan, respectively, and heme is in pink in CYP46A1 and in light pink in CYP2R1.

Fig. 3.

Comparison of the CH-3S-bound and ligand-free CYP46A1 structures. (a) Superposition of the two structures. The CH-3S-bound structure is colored in cyan, heme is in pink, and CH-3S is yellow except for the sulfate group, which is in orange. The ligand-free structure is colored in gray, and heme is in light pink. (b) Solvent-accessible surface of the ligand-free (in gray) and CH-3S-bound (in yellow) active sites. The volume does not change significantly from 309 ų in the ligand-free structure to 320 ų in the CH-3S-bound structure as calculated by VOIDOO (27). The active-site residues are colored in gray in the ligand-free structure and in cyan in CH-3S-bound. Side chains in contact with the steroid nucleus shift 0.6–4.2 Å upon substrate binding, whereas residues interacting with the sulfate group shift up to 9–12 Å in the two structures. For clarity, part of the B′–C loop region (residues 113–120) is not displayed.