Analysis of cytochrome P450 CYP119 ligand-dependent conformational dynamics by two-dimensional NMR and X-ray crystallography

Abstract

Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. We used two-dimensional (1)H,(15)N HSQC chemical shift perturbation mapping of (15)N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop with various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. The results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states.

Keywords: Conformational Change; Cytochrome P450; Molecular Dynamics; Nuclear Magnetic Resonance (NMR); X-ray Crystallography.

FIGURE 1.

Structures of azole inhibitors examined in this study: imidazole (A), 1-butylimidazole (B), 4-t-butylimidazole (C), 4-PI (D), 4-FPI (E), 4-CPI (F), 4-BPI (G), 2-BPI (H), 1-PI (I), econazole (J), and ketoconazole (K).

FIGURE 2.

¹H,¹⁵N HSQC overlap spectra of ferric ligand-free (blue) and Fe(II)-CO (red) of ¹⁵N-Phe-labeled CYP119 (A) and Fe(II)-CO (red) and Fe(II)-F310Y-CO (black) ¹⁵N-Phe-labeled CYP119 (B).

FIGURE 3.

Superimposition of the NMR spectrum of ¹⁵N-Phe-labeled wild-type CYP119 (blue) with the indicated ¹⁵N-Phe-labeled mutant (red) as follows: A, F5L; B, F24L; C, F36L; D, F39L; E, F60L; F, F87L; G, F98L; H, F144L; I, F153L.

FIGURE 4.

A, ligand-free (pink, PDB code, 1IO7), imidazole-bound (yellow, PDB code, 1F4U), and 4-PI-bound (cyan, PDB code, 1F4T) CYP119 aligned by using the heme moiety (gray spheres) as a reference. Selected Phe residues are represented as sticks. Residues of interest are labeled. The ligands are not shown. B, representation in A is rotated counter-clockwise, and the imidazole and 4-PI ligands are shown as yellow and cyan sticks, respectively.

FIGURE 5.

Superimposition of the NMR spectrum of ¹⁵N-Phe-labeled wild-type CYP119 (blue) with the indicated ¹⁵N-Phe-labeled mutant (red) as follows: A, F162Y; B, F225L; C, F228L; D, F292L; E, F298L; F, F310Y; G, F334L; and H, F338Y.

FIGURE 6.

Stereoview of substrate-free CYP119 (PDB code 1IO7). Phenylalanines are shown as black sticks surrounded by pink dots representing the van der Waals radii. For emphasis, the F, G, and I helices as well as the F-G loop have been drawn in color, whereas the remaining molecule is colorless. Phe-87 is behind the heme group.

FIGURE 7.

Titration of ¹⁵N-Phe-labeled CYP119 with lauric acid and stearic acid. ¹H,¹⁵N HSQC spectra monitored at 0.5 (red) and 1 (green) molar eq of lauric acid (A) and 1 (red) and 2 (green) molar eq of stearic acid superimposed upon the ligand-free protein spectrum (blue) (B).

FIGURE 8.

Superimposition of the NMR spectrum of ¹⁵N-Phe-labeled CYP119 (blue) with lauric acid-bound ¹⁵N-Phe-labeled (A) F87L, (B) F162Y, and F310Y CYP119, all in red (C).

FIGURE 9.

Superimposition of the NMR spectrum of 4-PI-bound ¹⁵N-Phe-labeled CYP119 (blue) with those of the following ¹⁵N-Phe-labeled mutants (red) as follows: A, F39L; B, F87L; C, F144L; D, F153L; E, F162Y; F, F225L; G, F228L; H, F310Y; and I, F338Y.

FIGURE 10.

¹H,¹⁵N HSQC spectrum of a 1 molar eq of each of the following ligands (red) superimposed on the spectrum of the ligand-free protein (blue): A, 4-PI; B, 4-FPI; C, 4-CPI; D, 4-BPI; E, 2-BPI; F, 1-PI; G, 1-butylimidazole; H, 4-t-butylimidazole; and I, imidazole.

FIGURE 11.

¹H,¹⁵N HSQC spectrum of a 1-molar eq of each of the following ligands (red) superimposed on the ligand-free protein spectrum (blue): A, econazole; B, ketoconazole.

FIGURE 12.

F and G helices of 4-PI-bound CYP119 (cyan) overlaid with the 4-BPI-bound CYP119 helices (green) using the heme as a reference (gray spheres). Selected residues and a portion of the backbone running through Gly-156 are drawn as sticks. For clarity, the 4-PI-bound F and G backbone has been represented as semi-transparent. Red arrows denote the point at which 4-(4-bromophenyl)-1H-imidazole-bound CYP119 shows the greatest deviation from the phenyl imidazole-bound structure.

FIGURE 13.

Overlay of the 4-BPI-bound (green) CYP119 structure with the 4-PI-bound (cyan) CYP119 structure.

FIGURE 14.

Simulated annealing F_o − F_c omit electron density maps surrounding ligands are shown in green. The F_o − F_c map was contoured to 2 σ. Ligands for 4-FPI (A), 4CPI (B), 4-BPI (C), and selected Phe residues are drawn as sticks. The heme moiety is represented by spheres. A portion of the protein backbone, including Gly-156, has been drawn as sticks. D, three halogenated ligands, 4-FPI (orange), 4-CPI (blue), and 4-BPI (green), are aligned using the heme moiety as a reference.

FIGURE 15.

¹H,¹⁵N HSQC spectra of ¹⁵N-Phe-labeled CYP119 with lauric acid (red) superimposed on those of the imidazole-bound (A) and 4-PI-bound (B) proteins (blue).