Human cytochrome P450 2E1 structures with fatty acid analogs reveal a previously unobserved binding mode

Abstract

Human microsomal cytochrome P450 (CYP) 2E1 is widely known for its ability to oxidize >70 different, mostly compact, low molecular weight drugs and other xenobiotic compounds. In addition CYP2E1 oxidizes much larger C9-C20 fatty acids that can serve as endogenous signaling molecules. Previously structures of CYP2E1 with small molecules revealed a small, compact CYP2E1 active site, which would be insufficient to accommodate medium and long chain fatty acids without conformational changes in the protein. In the current work we have determined how CYP2E1 can accommodate a series of fatty acid analogs by cocrystallizing CYP2E1 with omega-imidazolyl-octanoic fatty acid, omega-imidazolyl-decanoic fatty acid, and omega-imidazolyl-dodecanoic fatty acid. In each structure direct coordination of the imidazole nitrogen to the heme iron mimics the position required for native fatty acid substrates to yield the omega-1 hydroxylated metabolites that predominate experimentally. In each case rotation of a single Phe(298) side chain merges the active site with an adjacent void, significantly altering the active site size and topology to accommodate fatty acids. The binding of these fatty acid ligands is directly opposite the channel to the protein surface and the binding observed for fatty acids in the bacterial cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium. Instead of the BM3-like binding mode in the CYP2E1 channel, these structures reveal interactions between the fatty acid carboxylates and several residues in the F, G, and B' helices at successive distances from the active site.

FIGURE 1.

Overall structure of CYP2E1 with ω-imidazolyl decanoic fatty acid. The polypeptide is colored from blue at the N terminus to red at the C terminus with the major helices labeled. Heme is shown as black spheres, whereas the ligand is shown as magenta sticks. All of the figures were generated with PyMOL.

FIGURE 2.

Comparisons of CYP2E1 complexes with indazole (yellow), ω-imidazolyl octanoic fatty acid (green), ω-imidazolyl decanoic fatty acid (cyan), and ω-imidazolyl dodecanoic fatty acid (magenta). A, superposition of all four complexes. B, CYP2E1 interactions with the carboxylate termini of imidazolyl-fatty acids via Asn²⁰⁶ (octanoic, green; decanoic, cyan) or via contacts with both the G helix backbone and His¹⁰⁹ (dodecanoic, magenta). C, total omit map of ω-imidazolyl octanoic fatty acid contoured at 0.9 σ. D, total omit map of ω-imidazolyl decanoic fatty acid contoured at 1.0 σ. E, total omit map of ω-imidazolyl dodecanoic fatty acid contoured at 0.9 σ.

FIGURE 3.

Comparison of CYP2E1 voids with indazole (A), ω-imidazolyl octanoic acid (B), ω-imidazolyl decanoic acid (C), and ω-imidazolyl dodecanoic acid (D).

FIGURE 4.

Stereo view of CYP2E1 with indazole (yellow sticks) and with ω-imidazolyl decanoic acid (magenta sticks) demonstrates that the primary difference between the two structures consists of the rotation of Phe²⁹⁸, which allows merging of the two spaces observed in the indazole structure.

FIGURE 5.

Comparison of fatty acid analog binding in CYP2E1 (cyan) and BM3 (rose). The ligand in CYP2E1 is ω-imidazolyl decanoic fatty acid (cyan sticks). The ligand in BM3 is ω-imidazoyl-dodecanoic-l-leucine (Protein Data Bank code 3BEN).

FIGURE 6.

Exit routes from CYP2E1·imidazolyl-decanoic fatty acid structure as computed by CAVER. A, the top ranked route from the active site is shown in yellow and follows the access channel, whereas the next most likely route is shown in magenta and follows the fatty acid-binding site. B, relationship of the CAVER exit routes to the putative membrane surface.