Structural Studies Of Xanthomonas Important Proteins XC1160 And XC1553

Xanthomonas campestris pv.Campestris(Xcc)causes black rot,a common vascular disease with V-shaped lesions,which leads to significant economic losses.This work conducted structural studies on the important proteins XC1160 and XC1553 from Xcc.XC1160 plays an important role in signal transduction,while XC1553 functions in the interaction between the pathogen and plants.XC1160 serves as a response regulator in a two-component system.The coding gene for XC1160 was cloned into the expression vector p RSFDuet-1 and expressed as native protein and Se-Met derivatives in Escherichia coli(E.coli).Then target proteins were purified by nickel affinity chromatography and size exclusion chromatography in tandem,and protein crystals were obtained by the hanging drop vapor diffusion method.X-ray diffraction data was collected at Shanghai Synchrotron Radiation Center and utilized for further structure deter mination.Comparing the structure of XC1160 with those of Pho B and Hup R reveals that these response regulators share a similar structure.The overall structure of XC1160 adopts a core parallel β-sheets(S)wrapped by severalα-helices(H).The putative phosphorylation site of XC1160,Asp59,locates on S3 with an orientation different from the corresponding a mino acid of PhoB and Hup R.While the phosphorylation site Asp55 in Hup R binds directly to Be F3-,Asp59 in XC1160 indirectly contacts SO42-through the hydrogen bonds mediated by Lys108 and a water molecule.This suggest a special way for XC1160 to maintain local structure stability.The type III effector XC1553 is a Fic enzyme.It uridylylates the key kinases BIK1 and RIPK in plant immune signaling pathways to suppress the plant immune response.In this study,we mainly explored the expression and purification of XC1553.(1)To solve the problem of poor protein solubility,XC1553 gene cloned in the p RSFDuet-1 vector was transformed into E.coli strain BL21(DE3)and yielded highest expression at OD600= 1.5.(2)To further solve the problem of poor protein stability,mutations,truncations and co-expression(with PBL2)studies were performed for XC1553.(3)We expressed 9 homologous proteins of XC1553 and got 9 optimal constructs.Moreover,by using Biotin-UTP as the substrate,we established an enzyme activity assay to test whether prokaryotic expressed variants of XC1553 possess self-uridylylation activity.Notably,this assay obtains positive results for XC1553,Dscr2,Dscr5 and Dscr6.Differential scanning fluorescence method was used to analysis thermal stability of the homologous protein in the presence of UTP.Notably,AC4-1,AC4-2 and AC7-1 displayed increased thermal stability in UTP binding.The crystal screen for variants and homologs of XC1553 with good behavior in purification is on-going.This study reports the crystal structure deter mination of XC1160 and analysis of its putative phosphorylation site,which provides a new template and direction for structural research on response regulators.The problem in XC1553 expression lies in its solubility and stability.We found a solution to obtain large amounts of XC1553,by analyzing the expression and purification of its mutants,truncations,co-expressed complexes and homologous proteins,and selected better clones for further crystal screening.We established an enzymatic assay for detecting the activity of XC1553 which laid a solid foundation for further structural and biochemical research.