Interaction Mechanism Of Threonyl-tRNA Synthetase Of Phytophthora Sojae And Borrelidin Based On Molecular Simulation

Phytophthora sojae is a kind of oomycete,which causes soybean blight that leads different degrees of decay in leguminous plants.The prevention and control of soybean blight is still a global issue.Although the use of chemical agents such as metalaxyl is the common strategy to control of soybean blight,new anti-ovital drugs is more and more urgent due to the fact that the single action site of metalaxyl may cause serious resistance.Borrelidin?BN?,a natural product of microorganisms,is a macrolide compound targeting threonyl tRNA synthetase?ThrRS?and has antibacterial,anti-malarial and anti-cancer activities.However,it is also very toxic to human cells,indicating that the molecular structure of BN is still flawed and remains to be further optimized and reformed.Previous studies in our laboratory showed that BN has a unique high inhibitory activity against oomycetes including Phytophthora sojae.Its target is ThrRS of Phytophthora sojae?PsThrRS?,but the specific mechanism of action is not clear.In order to clarify the mechanism of interaction between BN and PsThrRS,which may provide reference information to drug design and structural modification of BN based on ThrRS as a target,this study uses homologous modeling method to obtain the three-dimensional structure model of wild-type PsThrRS and mutants using human ThrRS crystal structure as a template?Truncated PsThrRS after truncating the edit area?,followed by molecular docking and dynamic simulation with BN,respectively,to obtain the amino acid residue composition of the PsThrRS active center,and to compare the binding free energy of the truncated PsThrRS and BN docking of the wild type and the mutant type.At the same time,the key amino acid residues that interact with BN were clearly identified by point mutations,and a virtual screening of the compound database was sunsequnently conducted to provide a theoretical basis for obtaining potential anti-ocell drugs.The main results of this experiment are as follows:?1?The amino acid sequence of PsThrRS?GenBank:JF727824.1?and human TruRS crystal structure?PDB code:4HWT,4P3N,4TTV?were firstly obtained from NCBI database and PDB database,respectively,combined with molecular simulation technology in bioinformatics such as modeling have constructed a three-dimensional structural model of truncated PsThrRS.Molecular docking and kinetic simulations were used to summarize the 18 key amino acid residues that constitute the BN binding pocket in the wild-type PsThrRS active center:Ser-410,Gly-411,His-412,His-415,Tyr-416,Met-435,Cys-437,Arg-466,Gln-484,Phe-565,Tyr-566,Thr-586,Gln-588,Asp-590,Gln-592,Leu-593,His-616,Ala-618,in which 15 amino acid residues are conserved in prokaryotes and eukaryotes:Gly-411,His-412,Tyr-416,Met-435,Cys-437,Arg-466,Gln-484,Phe-565,Tyr-566,Thr-586,Gln-588,Asp-590,Leu-593,His-616,Ala-618.In addition,the results of kinetic simulations show that there are 6 additional amino acids in the PsThrRS that interact with BN:Pro-438,Arg-478,Phe-482,His-488,Ala-564,and Gly-621.Molecular docking results showed that Arg-466 formed a double hydrogen bond with O31,O32 of BN-C30 during the interaction,Gln-484 formed a single hydrogen bond with O32 on BN-C30,and Tyr-566 formed a single hydrogen bond with the hydroxyl group of BN-C25.Tyr-566 and BN-C13 form a covalent double bond consisting of a?bond and a?bond,and Asp-590 forms a single hydrogen bond with the hydroxyl group on BN-C13.Thus,BN is stably fixed in the binding pocket,demonstrating the deep fit of BN and PsThrRS.The mutants PsThrRS-R466A,PsThrRS-R478A,PsThrRS-Y566A,and PsThrRS-G621A were constructed by the virtual mutation technique and then docked with BN,however,the decreases with values of 0.1-1.35 kcal/mol were observed for the binding energy.The results showed that amino acid residues Arg-466,Arg-478,Tyr-566 and Gly-621 on PsThrRS may play a key role in the binding of BN to PsThrRS and the catalysis of substrates.?2?Three mutant expression vectors PsThrRS-R466A,PsThrRS-R478A and PsThrRS-Y566A were successfully constraucted by point mutation technique and overlap PCR.These three mutant PsThrRS expression vectors together with the wild-type PsThrRS expression vector were then individually transformed to E.coli BL21?DE3?.The recombinant protein was induced to express and purified.Through SDS-PAGE analysis,the optimal expression level was obtained when the recombinant strain was at a final concentration of 0.2 mmol/L at 16°C and the recombinant protein was mainly in a soluble form,the presence of inclusion bodies was less;the supernatants were subjected to nickel column gradient elution,ultrafiltration,concentration,and other purification treatments.It was found that the contaminants could be eluted after elution at 70 mM imidazole concentration.After elution with 100 mM imidazole concentration eluate,the target protein can be eluted,and the recombinant PsThrRS with a purity of more than 95%and a concentration of 1 mg/mL can be obtained finally.?3?After determination of the basic enzyme kinetic parameters of wild-type PsThrRS,combined with the Mie equation curve,the ATP K_m=32.09?M,V_max=4.62?M/min,K_cat=6.32min^-1;Ser K_m=0.09 mM,V_max=4.25?M/min,K_cat=5.82 min^-1;tRNA K_m=2.68?g/100?L,V_max=4.07?M/min,K_cat=5.58 min^-1,PsThrRS has greater affinity for substrate ATP than Ser.At the same time,the enzyme activities of PsThrRS mutants were measured and compared with their wild type PsThrRS.The results showed that the activity of mutant PsThrRS-R466A,PsThrRS-R478A and PsThrRS-Y566A decreased by 20.22%,57.80%and 58.84%,respectively.Therefore,it can be speculated that the amino acid residues R466,R478 and Y566 are related to the catalysis of the substrate in the aminoacylation reaction,and the accuracy of the molecular simulation results was verified experimentally.?4?The IC₅₀ of BN against wild-type PsThrRS was 4.4 nM.The IC₅₀ of the inhibitory mutant PsThrRS-R466A,PsThrRS-R478A and PsThrRS-Y566A were 4.6?M,1.4?M and 2.6?M,respectively.The concentration of enzyme in this study system was 0.73?M,compared to the wild type PsThrRS apparent inhibition constant K_i^?app?value increased 300-1000 times.Thus,mutations in Arg-466,Arg-478,and Tyr-566 can cause PsThrRS to produce significant resistance to BN.These reults also verifies the credibility of the conclusion that the binding energy reduces after the virtual point mutation results.?5?Thirty-seven small molecule compounds with a BN structure similarity of more than 70%were ontained from CHEMBL databases.Using the molecular docking software AutoDock vina for virtual screening,30 lead compounds with free binding energy lower than that of BN and affinity with PsThrRS were obtained.Finally,three selected compounds that have the best docking and potential for controlling P.sojae:CHEMBL2349167,CHEMBL2349177,and CHEMBL2349166 were obtained.Compared with BN,O31 of the carboxyl group located on C2 of the five-membered ring in the three compounds is replaced by tyrosyl,benzyl,and alanyl,respectively,indicating that Arg-466,which forms a double hydrogen bond with the carboxyl group on C2,is a key amino acid for PsThrRS.The above results provide new references and theoretical guidance for the development of new antifungal drugs.