Structural Basis Of The Carboxy-terminal Domain From Mps1 Regulating The Interaction With Itself And Other Proteins In Magnaporthe Oryzae

Magnaporthe oryzae,the fungal pathogen causing rice blast disease worldwide,is a model fungus for studying plant-pathogen interactions.Mps1 located at the most downstream of MoMck1-MoMkk1-Mps1 signaling transduction pathway,which is required for appressorium penetration and in plant invasive growth of M.oryzae.Mps1 regulates the pathogenesis through phosphorylation at its TXY residues by upstream MAPKKs.The phosphorylation of Mps1 has been regulated by the upstream kinase MoMkk1 and the protein phosphatase Pmp1.However,the crystal structure of Mps 1 and its homologs in fungi have not been reported,and the interaction mechanism of Mps1 with the upstream kinase kinase(MoMkk1)and the regulatory mechanism of Mps1 with phosphatase(Pmp1)remain unknown.Structural biology of Mps1 was studied,and main research results are as following:Firstly,the recombinant expression and purification of Mps1 were carried out.Large amounts of stable Mps1 protein with high purity and homogeneity were obtained.Crystal screening and optimization were performed using the sitting-drop vapor-diffusion method and crystals suitable for X-ray diffraction were obtained.Several sets of diffraction data with high resolution were collected and then the structure of Mps1 was solved to 1.99 A resolution by molecular replacement using a model of the ERK2 structure as template.Secondly,these data revealed that Mps1 adopt a novel self-interaction model:The C-terminal tail of molecule A interacts directly with the MAP kinase domain of molecule B.Yeast two-hybrid and co-immunoprecipitation analyses further confirmed this self-interaction between the C-terminal tail(Mps1361-415)and the MAP kinase domain(Mps11-360)in vitro or in vivo.These results further indicated that the interactions between the C-terminal tail(Mps1361-415)and the MAP kinase domain(Mps11-360)were confirmed by gel filtration chromatography and analytical ultracentrifugation.Thirdly,yeast two-hybrid analysis indicated that the C-terminal tail of Mps1 modulates itself interaction with its upstream kinase MoMkk1 and the interaction phosphatase Pmp1.Meanwhile,further results indicated that the unique C-terminal tail(Mps1401-415)of Mps1 negatively regulates its phosphorylation(Y188)in Escherichia coli.Moreover,differential scanning fluorimetry screening analysis revealed that eight MAP kinase-inhibitors may interact with Mps1.And then,co-crystallization of Mps1 with these candidate MAP kinase-inhibitors was performed.While several crystals were obtained and diffracted,none of these candidate MAP kinase-inhibitors could be found in the final models.Preparation of the complex samples,screening of the crystal growth conditions and screening of new potential inhibitors are ongoing.In summary,this is the first solved the crystal structure of MAP kinases in plant pathogenic fungi.This study reveals a novel self-interaction mode of MAP kinases and provides some structural clues for further understanding the molecular mechanism of the self-interactions of the MAP kinases from Bckl-Mkkl-Slt2 MAP kinase singling pathway in fungi.And the results further provided the molecular mechanism of the MAP kinases(Mps1)interactions with their partners.Meanwhile,the structure determination of Mps1 with high resolution also lays a solid foundation for the structure-based green agro-chemical design targeted against Mps1.