Structural characterization of human cholesterol 7α-hydroxylase

Abstract

Hepatic conversion to bile acids is a major elimination route for cholesterol in mammals. CYP7A1 catalyzes the first and rate-limiting step in classic bile acid biosynthesis, converting cholesterol to 7α-hydroxycholesterol. To identify the structural determinants that govern the stereospecific hydroxylation of cholesterol, we solved the crystal structure of CYP7A1 in the ligand-free state. The structure-based mutation T104L in the B' helix, corresponding to the nonpolar residue of CYP7B1, was used to obtain crystals of complexes with cholest-4-en-3-one and with cholesterol oxidation product 7-ketocholesterol (7KCh). The structures reveal a motif of residues that promote cholest-4-en-3-one binding parallel to the heme, thus positioning the C7 atom for hydroxylation. Additional regions of the binding cavity (most distant from the access channel) are involved to accommodate the elongated conformation of the aliphatic side chain. Structural complex with 7KCh shows an active site rigidity and provides an explanation for its inhibitory effect. Based on our previously published data, we proposed a model of cholesterol abstraction from the membrane by CYP7A1 for metabolism. CYP7A1 structural data provide a molecular basis for understanding of the diversity of 7α-hydroxylases, on the one hand, and cholesterol-metabolizing enzymes adapted for their specific activity, on the other hand.

Keywords: X-ray crystallography CYP7A1; cytochrome P450; oxysterols.

Fig. 1.

CYP7A1 structure. A: Overall structure of CYP7A1 rainbow-colored from blue N terminus to the red C terminus. B: A sequence alignment and superposition of I helix in CYP7A1 (yellow), CYP19A1 (green, PDB code 3EQM), human CYP51A1 (blue, PDB code 3LD6), Mycobacterium tuberculosis CYP51 (pink, PDB code 1EA1) highlighting active site residue Asn289. C: CYP7A1 bound to cholest-4-en-3-one. Cholest-4-en-3-one (orange) is surrounded by 16 residues within 4 Å from different structural elements: B’ helix (H101, T104L, S105), B-C loop (I114), C helix (I125 and F129), H helix (R260), I helix (V281, W284, A285, and N289), the loop between the K helix and β1–4 strand (S360 and L361), and the loop between two β4 strands at the C terminus (G485, L486, and G487).

Fig. 2.

A: Superposition of 7KCh-bound (cyan) with cholest-4-en-3-one-bound (brown) structure. Labeled regions show displacement and different conformation (A’ helix). B: 7KCh binding. Selected residues, heme (salmon), and hydrogen bonding of 3β-OH and 7-keto groups of 7KCh (blue) are shown.

Fig. 3.

A model of cholesterol abstraction from the membrane by CYP7A1 (surface representation). Several cross-sections of the active site and access channel are shown.