Structural and thermodynamic consequences of 1-(4-chlorophenyl)imidazole binding to cytochrome P450 2B4

Abstract

The crystal structure of P450 2B4 bound with 1-(4-chlorophenyl)imidazole (1-CPI) has been determined to delineate the structural basis for the observed differences in binding affinity and thermodynamics relative to 4-(4-chlorophenyl)imidazole (4-CPI). Compared with the previously reported 4-CPI complex, there is a shift in the 1-CPI complex of the protein backbone in helices F and I, repositioning the side chains of Phe-206, Phe-297, and Glu-301, and leading to significant reshaping of the active site. Phe-206 and Phe-297 exchange positions, with Phe-206 becoming a ligand-contact residue, while Glu-301, rather than hydrogen bonding to the ligand, flips away from the active site and interacts with His-172. As a result the active site volume expands from 200 A3 in the 4-CPI complex to 280 A3 in the 1-CPI complex. Based on the two structures, it was predicted that a Phe-206-->Ala substitution would alter 1-CPI but not 4-CPI binding. Isothermal titration calorimetry experiments indicated that this substitution had no effect on the thermodynamic signature of 4-CPI binding to 2B4. In contrast, relative to wild-type 1-CPI binding to F206A showed significantly less favorable entropy but more favorable enthalpy. This result is consistent with loss of the aromatic side chain and possible ordering of water molecules, now able to interact with Glu-301 and exposed residues in the I-helix. Hence, thermodynamic measurements support the active site rearrangement observed in the crystal structure of the 1-CPI complex and illustrate the malleability of the active site with the fine-tuning of residue orientations and thermodynamic signatures.

Figure 1

The chemical structures of 1-CPI and 4-CPI. The numbering of nitrogen atoms is indicated.

Figure 2

(A) Divergent stereo view of the seven copies of 2B4/1-CPI in the asymmetric unit. The seven molecules are shown as ribbon diagrams and colored red (molecule A), orange (B), yellow (C), green (D), cyan (E), blue (F), and purple (G), respectively. The view is along the 3-fold axis of the hexamer. (B) Side view of the hexamer. The color scheme is the same as in (A), and N-termini are indicated. (C) Divergent stereo view of hydrophobic interactions around the 3-fold axis of trimer ABC. Molecules A, B, and C are colored red, orange, yellow, respectively. The side chains of F212, V216, F220, F223, L224, and F227 are shown as sticks. These residues and helices F and G are labeled in molecule A.

Figure 3

Divergent stereo view of the 2B4/1-CPI complex (molecule A). Helices and strands are colored yellow and blue, respectively. The termini and major helices are labeled. Heme and 1-CPI are shown as red and forest sticks, respectively.

Figure 4

Electron density map around 1-CPI in the active site of molecule A (A), which has strong density for 1-CPI, and molecule G (B), which has weak density for 1-CPI. The σA-weighted 2|Fo| - |Fc| map is contoured at 1σ and shown as blue mesh around the 1-CPI ligand and adjacent residues, and the heme. F206, F297, and 1CPI are labeled. The nitrogen atoms (N1 and N3) of the imidazole are indicated. (C) Divergent stereo view of 1-CPI in the active site of molecule A. Side chains of residues within a generous 5 Å contact distance from 1-CPI are shown as sticks and colored by elements (yellow carbon, red oxygen, and blue nitrogen). The carbon atoms of 1-CPI are colored gray, and the chlorine is colored cyan. The heme is shown as red sticks.

Figure 5

Divergent stereo views for superposition of the 2B4/1-CPI and 2B4/4-CPI structures. (A) The 2B4/4-CPI structure superposed onto the 2B4/1-CPI (molecule A). Side chains of residues within a generous 5 Å contact distance from the ligands are shown as sticks and colored by elements (orange/yellow carbon in 2B4/4-CPI or 2B4/1-CPI, red oxygen, blue nitrogen, and cyan chlorine). The carbon atoms of 1-CPI are colored gray. Heme is shown as red (2B4/1-CPI) or orange (2B4/4-CPI) sticks. Helices F and I are labeled. (B) Overlay of 2B4/4-CPI and 7 molecules of 2B4/1-CPI in the asymmetric unit. The 2B4/4-CPI structure is colored black. The seven molecules of 2B4/1-CPI are colored red, orange, yellow, green, cyan, blue, and purple, respectively. (C) Heme coordination in 2B4/1-CPI (molecule A) and 2B4/4-CPI. Heme and side chains of residues involved in coordination of heme propionates are shown as sticks and colored by elements (yellow carbon, red oxygen, and blue nitrogen) in 2B4/1-CPI. These residues in the 2B4/4-CPI structure are also shown, and their carbon atoms are colored orange. The iron is displayed as a red sphere.

Figure 6

ITC and spectral studies of 4-CPI (A) and 1-CPI (B) binding to the F206A mutant. The upper panels show the enthalpy changes, and the lower panels are the resulting integrated enthalpy data fit to a single binding site model. The equilibrium difference spectra data are shown in the upper panels (Inset-a). The spectral data were fit to the “tight binding” equation, yielding the binding curve shown in the lower panels (Inset-b). (C) Thermodynamic signatures of 4-CPI and 1-CPI binding to 2B4dH(H226Y) (10) (solid bars) and F206A (hatched bars) are compared. Binding free energy (ΔG), green; change in enthalpy (ΔH), blue; change in entropy (−TΔS), red. (D) The network of 8 hydrogen bonds in the 2B4/4-CPI structure. 4-CPI, Leu-362, Asn-479, and the side chains of Glu-301, Thr-302, and Thr-305 are shown as sticks and colored by elements (orange carbon, red oxygen, blue nitrogen, and cyan chlorine). Two waters are shown as red spheres. Hydrogen bonds are indicated by dashed lines. This network of hydrogen bonds must be diminished in the 2B4/1-CPI complex, because the new position of the Phe-206 side chain, which is shown as yellow sticks, requires the displacement of two water molecules. The van der Waals radius of the Phe-206 side chain is indicated by small dots.