Structural basis of drug binding to CYP46A1, an enzyme that controls cholesterol turnover in the brain

Abstract

Cytochrome P450 46A1 (CYP46A1) initiates the major pathway of cholesterol elimination from the brain and thereby controls cholesterol turnover in this organ. We determined x-ray crystal structures of CYP46A1 in complex with four structurally distinct pharmaceuticals; antidepressant tranylcypromine (2.15 Å), anticonvulsant thioperamide (1.65 Å), antifungal voriconazole (2.35 Å), and antifungal clotrimazole (2.50 Å). All four drugs are nitrogen-containing compounds that have nanomolar affinity for CYP46A1 in vitro yet differ in size, shape, hydrophobicity, and type of the nitrogen ligand. Structures of the co-complexes demonstrate that each drug binds in a single orientation to the active site with tranylcypromine, thioperamide, and voriconazole coordinating the heme iron via their nitrogen atoms and clotrimazole being at a 4 Å distance from the heme iron. We show here that clotrimazole is also a substrate for CYP46A1. High affinity for CYP46A1 is determined by a set of specific interactions, some of which were further investigated by solution studies using structural analogs of the drugs and the T306A CYP46A1 mutant. Collectively, our results reveal how diverse inhibitors can be accommodated in the CYP46A1 active site and provide an explanation for the observed differences in the drug-induced spectral response. Co-complexes with tranylcypromine, thioperamide, and voriconazole represent the first structural characterization of the drug binding to a P450 enzyme.

Figures

FIGURE 1.

Chemical structures of the CYP46A1 substrate cholesterol and compounds tested in the present study.

FIGURE 2.

Absolute and difference (insets) spectra of CYP46A1 in the absence (A) and presence of different drugs (B, TCP; C, THP; D, VOR; E, CLO). The concentration of CYP46A1 is 0.3 μm, and that of the ligand is 50 μm. The black line shows the CYP46A1 spectrum at 18 °C, and red line shows the spectrum at 37 °C.

FIGURE 3.

Unbiased composite omit electron density contoured at 1σ and 3σ for four CYP46A1 complexes: THP, TCP, VOR, CLO. For TCP and CLO, copy A of the asymmetric unit is shown; for VOR, copy B of the asymmetric unit is shown. For the THP and TCP complexes a portion of the electron density peak on iron is also present.

FIGURE 4.

Views of the CYP46A1 active site illustrating interactions with TCP. A, residues in contact with TCP are shown. Portions of the secondary structure are also shown. B, superposition of TCP-bound (slate) versus ligand-free (wheat) CYP46A1 show the active site volumes and amino acid residues that undergo conformational changes upon TCP binding. The heme group in TCP and ligand-free CYP46A1 is in light orange and red, respectively, and TCP is in yellow. The nitrogen, oxygen, sulfur, and iron atoms are in blue, red, yellow, and orange, respectively. H2O molecule 290 is shown as a red sphere. Dashed black lines indicate hydrogen bonds. Protein side chains are shown as sticks, and the solvent-occupied surface of the active site is shown as a light blue mesh (TCP-bound CYP46A1) and wheat surface (ligand-free CYP46A1).

FIGURE 5.

Views of the CYP46A1 active site illustrating interactions with THP. A, residues in contact with THP are shown. B, superposition of THP-bound (cyan) versus C3S-bound (magenta) CYP46A1, and (C) THP-bound (cyan) versus ligand-free (wheat) P450 show the position of the ligands and major conformational differences between the structures. The heme group in THP and C3S-bound co-complexes is in light pink and red, respectively, and in ligand-free CYP46A1 is in hot pink. THP is in lemon, and C3S is in light orange. The nitrogen, oxygen, sulfur, and iron atoms are in blue, red, yellow, and orange, respectively. H2O molecules are shown as red spheres. Dashed black lines indicate hydrogen bonds. The solvent occupied surface of the active site in THP-bound CYP46A1 is colored in gray.

FIGURE 6.

Views of the CYP46A1 active site illustrating interactions with VOR. A, shown are residues in contact with VOR and a network of bound H2O molecules. Portions of the secondary structure are also shown. B, superposition of VOR-bound (pale green) and ligand-free (wheat) structures show the active site volumes and conformational changes that accommodate binding of the VOR molecule. The heme group in VOR-bound and ligand-free CYP46A1 is in warm pink and red, respectively, and VOR is in light pink. The nitrogen, oxygen, iron, and fluorine atoms are in blue, red, orange, and light blue, respectively. H2O molecules are shown as red spheres. Dashed black lines indicate hydrogen bonds. The solvent-occupied surface of the active site is shown in green mesh in VOR-bound CYP46A1 and a wheat surface in ligand-free P450.

FIGURE 7.

Views of the CYP46A1 active site illustrating interactions with CLO. A, residues in contact with CLO are shown. B, superposition of CLO-bound (violet) and ligand-free (wheat) CYP46A1 structures shows the solvent-occupied surface of the active site in CLO-bound CYP46A1 and residues undergoing conformational changes upon CLO binding. The heme group in CLO-bound and ligand-free CYP46A1 is in salmon and red, respectively, and CLO is in gray. The nitrogen, oxygen, sulfur, iron, and chlorine atoms are in blue, red, yellow, orange, and green, respectively. H2O molecules are shown as red spheres. Dashed black lines indicate hydrogen bonds, and the dashed gray line connects the VOR nitrogen and heme iron showing the distance between the two atoms.

FIGURE 8.

Mass spectra of the extracts from the incubations of CYP46A1 with CLO at 25 °C (A) and 37 °C (B). C, the MS/MS of the peak at m/z 360.85 in B is shown.