Crystal Structure Studies Of Chuangxinmycin In Complex With Tryptophanyl-tRNA Synthetase

Objectives Using E.coli expression system to efficiently express Tryptophanyl-tRNA Synthetase(BsTrpRS)for co-crystallization with chuangxinmycin.In addition,solving the crystal structures of BsTrpRS in complex with chuangxinmycin will provide us more insight into the functions of chuangxinmycin in physiology and pharmacology,and also help us to design and optimize the chuangxinmycin-based drugs.Methods 1 The expression vector containing the target gene of BsTrpRS protein was constructed and the soluble expression of TrpRS protein was obtained by E.coli expression system.2 The target protein was purified by affinity chromatography,ion exchange chromatography and molecular size exclusion chromatography in order to obtain BsTrpRS,with purity of more than 90%.The activity of BsTrpRS was determined by surface plasmon resonance(SPR)and Isothermal Titration Calorimetry(ITC).3 High quality crystal for X-ray diffraction was obtained by optimizing crystallization conditions,including protein concentration,precipitant concentration,temperature and pH,by employing vapor diffusion suspension method.4 The crystal of Chuangxinmycin in complex with BsTrpRS was obtained by the method of co-crystallization.5 The crystal diffraction data were collected by X-ray diffraction,the corresponding complex structures were solved with Molecular Replacement(MR)method,and the molecular dynamics simulation and site-directed mutation verification were used to reveal the mechanism of the action of Chuangxinmycin on the BsTrpRS target.Results 1 The recombinant plasmid containing BsTrpRS gene was successfully constructed by molecular biological method,and the strain BL21(DE3)with high expression of BsTrpRS protein was obtained by optimization.2 High purity BsTrpRS protein was obtained by affinity chromatography,ion exchange chromatography and molecular size exclusion chromatography.3 The activity of the BsTrpRS was verified by the SPR and the ITC technique,and a high affinity between Chuangxinmycin and the BsTrpRS was confirmed.4 Through crystallization optimization and collection of X-ray diffraction data,the binary structure of Chuangxinmycin*BsTrpRS at a resolution of 2.06? and the ternary structure of Chuangxinmycin*ATP*BsTrpRS at a resolution of 2.16? were determined.Combining the crystal structures with molecular dynamics simulation and sitedirected mutation verification,the mechanism of the interaction between Chuangxinmycin and BsTrpRS was revealed at the molecular level.Conclusions This study elucidates the mechanism of the interaction between Chuangxinmycin and its target BsTrpRS at the molecular level,which provides a precise molecular structural model for the binding of Chuangxinmycin with BsTrpRS,and lays a theoretical foundation for later screening and rational design of more effective innovative drugs.Figure 34;Table 13;Reference 111...