Two-dimensional NMR and all-atom molecular dynamics of cytochrome P450 CYP119 reveal hidden conformational substates

Abstract

Cytochrome P450 enzymes are versatile catalysts involved in a wide variety of biological processes from hormonal regulation and antibiotic synthesis to drug metabolism. A hallmark of their versatility is their promiscuous nature, allowing them to recognize a wide variety of chemically diverse substrates. However, the molecular details of this promiscuity have remained elusive. Here, we have utilized two-dimensional heteronuclear single quantum coherence NMR spectroscopy to examine a series of mutants site-specific labeled with the unnatural amino acid, [(13)C]p-methoxyphenylalanine, in conjunction with all-atom molecular dynamics simulations to examine substrate and inhibitor binding to CYP119, a P450 from Sulfolobus acidocaldarius. The results suggest that tight binding hydrophobic ligands tend to lock the enzyme into a single conformational substate, whereas weak binding low affinity ligands bind loosely in the active site, resulting in a distribution of localized conformers. Furthermore, the molecular dynamics simulations suggest that the ligand-free enzyme samples ligand-bound conformations of the enzyme and, therefore, that ligand binding may proceed largely through a process of conformational selection rather than induced fit.

FIGURE 1.

Comparison of crystal structures for the ligand-free (A), imidazole-bound (B), and 4-phenylimidazole-bound (C) CYP119 enzyme.

FIGURE 2.

A, ¹H,¹³C-HSQC spectra of the ligand-free CYP119 [¹³C]MeOF mutants. B, ¹H,¹³C-HSQC spectra of the ligand-free CYP119-F162[¹³C]MeOF mutant after reduction with dithionite and binding to CO.

FIGURE 3.

¹H,¹³C-HSQC spectra of the ligand-bound CYP119 [¹³C]MeOF mutants F144[¹³C]MeOF, F153[¹³C]MeOF, and F162[¹³C]MeOF, respectively. For the sake of brevity, only the 1:1 (protein:ligand) stoichiometric concentration is shown. Ligands used are as follows: F144[¹³C]MeOF with imidazole (A), 4-phenylimidazole (D), and lauric acid (G); F153[¹³C]MeOF with imidazole (B), 4-phenylimidazole (E), and lauric acid (H); and F162[¹³C]MeOF with imidazole (C), 4-phenylimidazole (F), and lauric acid (I). Arrows point to ligand-bound resonances.

FIGURE 4.

Crystal structure of the ligand-free CYP119 enzyme color-coded to regions of dynamic motion as identified by the 200-ns all-atom molecular dynamics simulation. Blue regions indicate areas with the least amount of fluctuation from the ligand-free starting structure (<1 Å r.m.s.d.), whereas red regions indicate areas with the greatest degree of fluctuation from the ligand-free structure (>5 Å r.m.s.d.).

FIGURE 5.

A, the 66-ns time point from the all-atom molecular dynamics simulation (copper) superimposed upon the 4-phenylimidazole-bound CYP119 crystal structure (blue). B, close-up view of the F/G loop region (residues 151–159) showing the adoption of the ligand-bound conformation at ∼66 ns into the simulation.

FIGURE 6.

A, the 200-ns endpoint of the all-atom molecular dynamics simulation (yellow) superimposed upon the 4-phenylimidazole-bound CYP119 structure (blue). B, close-up view of the F/G loop region (residues 151–159) showing deviation from the ligand-bound conformation.

FIGURE 7.

Plot of the average r.m.s.d. of the F/G loop region (residues 151–159) of the ligand-free form of CYP119 versus the time course of the 200-ns molecular dynamics simulation. Each point corresponds to 2 ns of the 200-ns trajectory.