Investigation Of The Binding Mechanism Between α-TOH And CYP4F2 Using Molecular Dynamics Simulations

Cytochrome P450(CYP)are ubiquitous heme-containing monooxygenases that metabolize endogenous and exogenous substrates.The metabolic pathways include hydroxylation,dealkylation and epoxidation.CYP4 family is the second largest human CYP family.Among them,cytochrome P450 4F2(CYP4F2)is an ω-hydroxylase.CYP4F2 has multiple functions including 20-hydroxyeicosatetraenoic acid production,catabolism of leukotrienes and the long chain fatty acids,and the initiation of Vitamin K and Vitamin E catabolism.α-Tocopherol(α-TOH)is the most bioactive and bioavailable antioxidant in vitamin E and it is one of the main substrates of CYP4F2.Elucidating the binding mechanism of CYP with substrate is beneficial for understanding the structure-function relationships of CYP4F2 and provides valuable theoretical guidance for substrate-based drug design.In this paper,homology modeling was used to construct the structure of CYP4F2 and molecular docking was used to obtain the complex model of α-TOH with CYP4F2.Molecular dynamics simulations combined with multiple analysis methods were performed on complex systems to identify the binding mechanism of α-TOH with CYP4F2 and elucidate the influence of mutation on α-TOH-binding.The main contents of the paper are as follows:1.Molecular dynamics simulations of the interactions between CYP4F2 and α-TOHα-Tocopherol(α-TOH)is a potent antioxidant,which has the highest biological activity and bioavailability among the eight members of Vitamin E(α-,β-,γ-andδ-tocopherol and α-,β-,γ-and δ-tocotrienol).The plasma concentrations of α-TOH are related to human health.The insufficient concentrations of α-TOH are related to stunting of growth,anemia and increased infection.The high concentrations of α-TOH could be associated with many adverse effects.Cytochrome P450 4F2(CYP4F2)is the only P450 enzyme in the body that metabolizes α-TOH.CYP4F2 can regulate the concentrations of α-TOH in the body which activate the α-TOH metabolism byω-hydroxylating the terminal methyl group of α-TOH.Clarifying the details of the interactions between α-TOH with CYP4F2 are important to understand the metabolic process of α-TOH.Till now,the 3D structure of CYP4F2 has not been determined.In this thesis,we constructed the α-TOH-CYP4F2 complex model by homology modeling and molecular docking.Molecular dynamics simulations combined with multiple analysis methods were performed on complex systems to identify the key factors to the binding of α-TOH with CYP4F2.MM-GBSA method combined with free energy landscape show that the key factors to α-TOH binding are the hydrophobic interaction and hydrogen bond interaction.8 key residues(V67,V76,F124,V395,P396,V397,L421 and L504)establish a hydrophobic cavity stabilizing α-TOH in the pocket of CYP4F2 and S423 forms an important hydrogen bond with α-TOH anchoring α-TOH in the favorable position for ω-hydroxylation.Our results could enrich the information on the structural features of CYP4F2 and help us understand the interactions betweenα-TOH and CYP4F2,which provide valuable theoretical guidance for the study of CYP4F2-mediated ω-hydroxylation of other substrates.2.The effects of mutations in important residues of CYP4F2 on the binding of α-TOH with CYP4F2Based on the calculations of the previous part,combined with the relevant experimental data and the structural characteristics of CYP4F2,we speculate that S423,V433 and E328 are important for the binding of α-TOH to CYP4F2.In the previous study,we found that α-TOH establishes only a single hydrogen bond with the S423 residue directly.We can speculate S423 plays a crucial part in the binding of α-TOH with CYP4F2.Furthermore,it has been reported that V433 M mutation would decrease the ω-hydroxylation activity of α-TOH significantly.We noticed that the position of the V433 is situated far away from the active site of CYP4F2.How the distal residue V433 affects the structural characteristics and substrate binding of CYP4F2 inspires our interest.Moreover,the glutamic acid in I helix of CYP4 family could form an ester bond with heme,which enables the CYP4 to selectively hydroxylate the substrate atω-site.Residue E328 in CYP4F2 forms an ester bond with heme.The effect of the ester bond between E328 and heme on the hydroxylation of α-TOH remains unclear.To investigate the effects of S423,V433 and E328 on the binding of α-TOH with CYP4F2,we performed molecular dynamics simulations on S423 A,V433M and E328 A mutation systems respectively.The results show that the disappearance of hydrogen bond between S423 and α-TOH leads to the shift of α-TOH,weakens the interaction betweenα-TOH with CYP4F2 and destroys the distance condition for ω-hydroxylation.V433 remote regulates the binding of α-TOH with CYP4F2.The V433 M mutation causes the conformational change of CYP4F2,destroying the original hydrophobic cavity and the important hydrogen bond between S423 and α-TOH.The ester bond between E328 and heme restricts α-TOH to a narrow cavity,facilitating the ω-hydroxylation of α-TOH.In E328 A mutation system,the disappearance of ester bond increases the probability of contact between the C atom at the ω-1 site and heme,which increases the possibility of hydroxylation at the ω-1 site of α-TOH,and weakens the selectivity of CYP4F2 forω-hydroxylation of α-TOH.The mutations directly or indirectly change the binding mode of α-TOH and decrease its binding affinity with CYP4F2,which is unfavorable for ω-hydroxylation.This work expounds the effect of important residues mutations on the binding of α-TOH with CYP4F2 at the atomic level,and it is helpful to better understand the structure-function relationships of CYP4F2.