Structural Bioinformatics Studies Of CYP2E1 Binding With Aromatic Substrates

The cytochrome P450s (CYPs) have the closest relationship with drug metabolism in human. Among CYPs, CYP2E1 is in charge of 6% drug metabolism and metabolize a diversity of drugs with far distinct structures and chemical properties, such as alcohols, monocyclic compounds, bicyclic heterocycles and even fatty acids. And recent released crystal structure shows that CYP2E1 has the smallest pocket volume and there exists a 2nd small cavity near the active site other than the access channel entry, which is a rare finding in the CYP 450s family. It needs to be study why this enzyme could metabolize such diverse substrates within so limited volume. Furthermore, CYP2E1 also shows atypical steady-state kinetics for monocyclic substrates, which indicates negative cooperative binding with two substrates, leading to metabolic inhibition. However the mechanism of substrate diversity and negative cooperative binding is unclear. The thorough investigation was carried out by docking of five aromatic substrates (benzene, aniline, acetaminophen, chlorzoxazone and theophylline) different in structures and characteristics into X-ray structure of CYP2E1, then all-atom molecular dynamics simulations (MD) of 5 ns were performed to study the mechanism of substrate specificity in human CYP2E1. To further understanding the negative cooperative binding, aniline, as the aromatic monocyclic substrate with a minimum substitution, was selected to undergo 10 ns MD involving different models with one or two molecules binding to CYP2E1. The simulation results were analyzed carefully in aspects of RMS derivations, close contact interactions, etc. For the researching of negative binding, the covariance analysis and Gibbs free binding energy calculation were added, which gived us quiet fruitful feedbacks. Results show that the aromatic substrates interact with the active site mainly throughπ-πstacking with Phe106, Phe116, Phe207, Phe298 and Phe478, and almost free from hydrogen bond interactions. Different energetic clustering profiles and structural characteristics resulting from substrate movement play a key role in determining the product diversity. Molecules with single catalytic site such as benzene and chlorzoxazone always only have one conformation cluster. While substrates with multiple catalytic sites always adopt more diverse energetic clustering profiles and also behave more active in the binding pocket. However, the negative cooperative binding comes from the affection of the 2nd aniline on the active aniline through interacting with F478, a key residue in the linkage of access channel and active site. Also the additional binding significantly lowers the activity of T303, a key factor in the substrate positioning. All these findings provide an atomic insight into the mechanism of the substrate diversity and negative cooperative binding in human CYP2E1, which are useful for conducting mutagenesis studies, providing insights into personalization of drug treatments and stimulating novel strategies for finding desired personalized drugs.