Structures of human cytochrome P-450 2E1. Insights into the binding of inhibitors and both small molecular weight and fatty acid substrates

Abstract

Human microsomal cytochrome P-450 2E1 (CYP2E1) monooxygenates > 70 low molecular weight xenobiotic compounds, as well as much larger endogenous fatty acid signaling molecules such as arachidonic acid. In the process, CYP2E1 can generate toxic or carcinogenic compounds, as occurs with acetaminophen overdose, nitrosamines in cigarette smoke, and reactive oxygen species from uncoupled catalysis. Thus, the diverse roles that CYP2E1 has in normal physiology, toxicity, and drug metabolism are related to its ability to metabolize diverse classes of ligands, but the structural basis for this was previously unknown. Structures of human CYP2E1 have been solved to 2.2 angstroms for an indazole complex and 2.6 angstroms for a 4-methylpyrazole complex. Both inhibitors bind to the heme iron and hydrogen bond to Thr303 within the active site. Complementing its small molecular weight substrates, the hydrophobic CYP2E1 active site is the smallest yet observed for a human cytochrome P-450. The CYP2E1 active site also has two adjacent voids: one enclosed above the I helix and the other forming a channel to the protein surface. Minor repositioning of the Phe478 aromatic ring that separates the active site and access channel would allow the carboxylate of fatty acid substrates to interact with conserved 216QXXNN220 residues in the access channel while positioning the hydrocarbon terminus in the active site, consistent with experimentally observed omega-1 hydroxylation of saturated fatty acids. Thus, these structures provide insights into the ability of CYP2E1 to effectively bind and metabolize both small molecule substrates and fatty acids.

FIGURE 1.

Overall structure of CYP2E1 and overlay with CYP2A13. A, distal face of CYP2E1 rainbow colored from N terminus (blue) to C terminus (red). Helices are labeled using typical P-450 nomenclature. B, structural overlay of CYP2E1 (blue) and CYP2A13 (green) using secondary-structure matching (69) in COOT (31) with a Cα root mean square deviation of 1.00 Å. All of the protein structure figures were generated using PyMOL (70).

FIGURE 2.

Heme and ligand electron density maps. Electron density shown as composite omit σ_A-weighted 2|F_o| - |F_c| map at 1.0 σ around the heme and indazole (A) or 4-methylpyrazole (B).

FIGURE 3.

Active site of CYP2E1. Wall-eyed stereo views of the CYP2E1 active site illustrating constrictions between the ligand-containing active site, the small distal void, and the substrate access channel. Cavities are shown in gray mesh. Substrate access channel is omitted from A and F-helix was omitted from B for clarity. Helices and loops are colored as indicated: B′ helix and adjacent loop (blue); F helix (orange); G helix (purple); I helix (yellow); loop between helix K and β_1-4 (green); and β_4-1/β_4-2 turn (pink).

FIGURE 4.

CYP2E1 access channel location. A, access channel, active site, and extra volume are shown in black mesh. Protein regions bordering the access channel entrance are colored green. B, active site void connectivity using a 1.4 Å radius probe (black mesh) versus a 0.9 Å radius probe (green mesh). C, most accessible exit path calculated by CAVER (36) is shown as light gray spheres. Residues proposed to interact with fatty acids are shown in yellow.

FIGURE 5.

Active site and access channel comparisons for human xenobiotic-metabolizing cytochrome P-450 enzymes. A, CYP2E1; B, CYP2A6 (Protein Data Bank code 1Z10); C, CYP2A13 (Protein Data Bank code 2P85); D, CYP1A2 (Protein Data Bank code 2HI4); E, CYP2C9 (Protein Data Bank code 1OG2); F, CYP2C8 (Protein Data Bank code 1PQ2); G, CYP2D6 (Protein Data Bank code 2F9Q); H, CYP3A4 (Protein Data Bank code 1TQN). The volumes were calculated by VOIDOO as described under “Materials and Methods.”

FIGURE 6.

Lauric acid structure overlaid on CYP2E1, demonstrating the distance from the active site to the QXXNN access channel residues proposed to bind the carboxylate of fatty acid substrates. In this example lauric acid is interacting with Gln²¹⁶ and Asn²¹⁹, and the subterminal carbon is in position for the experimentally observed ω-1 hydroxylation.

FIGURE 7.

Electrostatic surface of CYP2E1. Electrostatic surface of CYP2E1 as calculated by APBS (71) showing the positively charged surface proposed to be the cytochrome b₅/NADPH-cytochome P-450 reductase-binding site. Arrows marking the location of helices are oriented along the long axes of helices to aid in spatial orientation. Heme shown as green spheres.