Molecular Dynamics Simulation Study On The Interaction Mechanism Of P450-substrates And Tcar-DNA

The physiological function of protein is inseparable from the changes of its conformation,bound with the substrates and interacted with the nucleic acid.Cytochrome P450 s are heme-containing monooxygenases and are involved in a series of redox reactions by transferring electrons.P450 s can not only metabolize a variety of endogenous,but also play an important role in the metabolism of exogenous,which can promote the detoxification and elimination of chemical compounds.Tca R,a member of Mar R family,can regulate the transcription of ica ADBC,spa,sas F and sar S.The latest research shows that Tca R can interact with ss DNA,which plays a vital role in interfering with ss DNA replication and resisting ss DNA phage invasion.In this dissertation,the structure and properties of P450 enzymes,as well as the recognition mechanism of Tca R-ss DNA were studied by molecular dynamics simulation.The content mainly included the following three parts:1.Molecular dynamics simulation studies of the substrate selectivity of CYP3A7.CYP3A7 is one of the CYP3 family members,which is very active in the fetal liver and also detectable in some adult liver and intestine.It carries out a similar role in fetuses that CYP3A4 serves in adults.CYP3A7 can oxidize the exogenous compounds in order to exclude them from body and play an important role in metabolized steroids.The present study has not yet provide the crystal structure of CYP3A7,the features of CYP3A7 remain unknown at the atomic level.In this study,a three-dimensional model of CYP3A7 was constructed by homology modeling.The docking method combined with molecular dynamics simulation and binding free energy were adopted,to probe the substrate(DHEA,estrone and estradiol)selectivity of CYP3A7.Moreover,several key residues responsible for substrate specificity were identified.We found that the dominant access channel for three substrate is channel S.This study provides knowledge of the substrate selectivity of CYP3A7,which could contribute to further understanding of related protein structures and dynamics.2.Exploring the Structure Characteristics and Major Channels of Cytochrome P450 2A6,2A13 and 2E1 with PilocarpineThe functional CYP2 A contains two enzymes,CYP2A6 and CYP2A13,which have 94% sequence similarity with each other.Both of CYP2A6 and CYP2A13 can metabolize many procarcinogens from tobacco.CYP2E1 belongs to the CYP2 family and has < 40% sequence similarity with CYP2 A enzymes.Although the active area of CYP2E1 is small,it can still metabolize exogenous substrates to produce toxic substances and carcinogens.Therefore,inhibition of activity of CYP2A6,CYP2A13 and CYP2E1 can promote the detoxification and elimination of chemical carcinogens.Three different P450 enzymes combined with the same inhibitor pilocarpine were chose to direct comparisons,to clarify the difference of three P450 enzymes inhibitory activity at the atomic level and analyze the dominant access channel for three P450 enzymes.We proposed a “gating mechanism” of their respective major channels for three enzymes,which may be attributed to a reversal of Phe209 in CYP2A6/CYP2A13,as well as the rotation of Phe116 and Phe298 in CYP2E1.The present study provides important insights into the structure-function relationships of three cytochrome P450 s and the molecular basis for development of potent inhibitors.3.Studying the recognition mechanism of Tca R and ss DNA usingmolecular dynamic simulationsThe transcription regulator teicoplanin-associate locus regulator(Tca R)is a member of the multiple antibiotic resistance regulator(Mar R)family.To date,a number of Mar R family members can bind with double-stranded DNA(ds DNA).Recently,studies demonstrated that Tca R could strongly interact with single-stranded DNA(ss DNA).Tca R-ss DNA interaction plays a key role in regulating DNA replication and resisting ss DNA phage invasion,which suggests a novel function for Tca R in bacteriophage resistance.However,the dynamical information and recognition mechanism of dimeric Tca R bound to ss DNA have not been rationalized at the atomic level.We selected apo-Tca R,Tca R-ss DNA and two quadruple mutants(MTA and MTB)as the research object to study recognition mechanism between Tca R and ss DNA.The results illuminate that electrostatic interaction is the main driving force during the binding process.We put forward that six anchoring residues(Arg70,Arg71,Ser188,Gln191,Arg221 and Arg222)play a vital role in stabilizing the ss DNA by forming strong hydrogen bond and salt bridge interactions.Tca R undergoes the asymmetric conformational changes at the w HTH domain upon binding to ss DNA.This may be attributed to the changing of electrostatic potential,enhanced contacts and salt bridge interaction.The present study provides new insights into the recognition mechanism of Tca R bound to ss DNA,which could contribute to understanding about the multiple Tca R functions in staphylococci enrich our understanding of Mar R family.