Comparison of the dynamics of substrate access channels in three cytochrome P450s reveals different opening mechanisms and a novel functional role for a buried arginine

Abstract

Understanding the mechanism and specificity of substrate binding in the cytochrome P450 (P450) superfamily is an important step toward explaining its key role in drug metabolism, toxicity, xenobiotic degradation, and several biosynthetic pathways. Here we investigate the ligand exit pathways and mechanisms of P450cam (CYP101), P450BM-3 (CYP102), and P450eryF (CYP107A1) by using random expulsion molecular dynamics and classical molecular dynamics simulations. Although several different pathways are found for each protein, one pathway is common to all three. The mechanism of ligand exit along this pathway is, however, quite different in the three different proteins. For P450cam, small backbone conformational changes, in combination with aromatic side chain rotation, allow for the passage of the rather rigid, compact, and hydrophobic substrate, camphor. In P450BM-3, larger transient backbone changes are observed on ligand exit. R47, situated at the entrance to the channel, appears important in guiding negatively charged fatty acid substrates in and out of the active site. In P450eryF, an isolated buried arginine, R185, stabilized by four hydrogen bonds to backbone carbonyl oxygen atoms, is located in the exit channel and is identified as having a particularly unusual functionality, dynamically gating channel opening. The results for these three P450s suggest that the channel opening mechanisms are adjusted to the physico-chemical properties of the substrate and can kinetically modulate protein-substrate specificity.

Crystal structures and catalytic mechanism of cytochrome P450 StaP that produces the indolocarbazole skeleton

“…The third CPA is located between helix BЈ1 and ␤-1,2. Although the functional significance of the latter two molecules is unclear, and the binding of these two additional molecules might represent a crystallographic artifact, the site between helix BЈ1 and ␤-1 is a plausible entry point for CPA because this region is flexible in the substrate-free form and has been proposed to be a substrate entry site for several P450s (16,17).…”

Slide-and-exchange mechanism for rapid and selective transport through the nuclear pore complex

“…To assess the predominance of sliding over other modes of motion, we complemented our Anton simulations with 5,400 independent and relatively short (150-1,500 ps) randomly accelerated molecular dynamics (RAMD) simulations (60) of a single FG motif at the NTF2 crystallographic binding site, for a total of 3,820 ns of simulations. In contrast to most enhanced sampling methods, RAMD has often been used to find favorable modes of motion of a ligand or structural element and avoid a priori assumptions about the direction of motion (61,62). Using RAMD, we iteratively applied a randomly oriented steering force of fixed magnitude to the FG motifs, setting the magnitude of the steering force between 5.0 and 12.5 kcal mol −1 Å −1 in different simulations.…”

The Cytochrome P450 3A4 has three Major Conformations: New Clues to Drug Recognition by this Promiscuous Enzyme

“…[29] The dynamic binding to the substrate suggests an entropy controlled desolvatation, which could be related to the substrate lipophilicity. [30] The p-p interaction was defined as a contributing factor in lipophilicity relations. [31] In fact, the statistical relation established on lipophilicity provides directly a measure of p-p interactions energies.…”