Structural Determination Of MoTctex1 And MoTctex1-ICp Complex And Analysis Of Complex Interaction Characteristics In Magnaporthe Oryzae

Rice blast fungus?Magnaporthe oryzae?is the pathogen that causes rice blast disease,which is one of the most devastating diseases,bringing about more than 10%rice yield reduction per year worldwide.Conducting studies on the growth and development of rice blast fungus is important for the understanding of the mechanism of control and regulation of rice blast fungus.Mycelium growth in M.oryzae involved in many important biological processes such as biosynthesis,transport and signal transconduct of growth and development related proteins.In the previous research,our data shown that deletion mutants of MGG₀1005 had a slower speed in Mycelium growth.and a low sequence homology with the known functional proteins in protein database.Here,we determine the structure of the hypothetical protein,MGG₀1005 and show that it is the Magnaporthe oryzae Dynein light chain Tctex-type 1,demonstrated by its structural similarity to other orthologous dynltl proteins and its conserved interaction with the Magnaporthe oryzae dynein intermediate chain,MoDIC2.These results demonstrate the utility of structure-based annotation and validate it as a viable approach for the molecular assignment of hypothetic proteins from phyto-pathogenic fungi.In addition,we present the structure of the MGG 01005-MoDICp complex together with functional studies of MGG₀1005.All the data have a good academic value for the drug target screening,and provide some theoretical and practical basis for the prevention and treatment of rice blast fungus.Expression vector and purification system have been established in this study.A large number of homogeneous proteins have been obtained.After crystal condition screening and optimization,the protein structure was determined by single wavelength anomalous scattering.The structure showed that MGG₀1005 was a homologous dimer.The monomer structure consisted of 2 a helices and 4 P-strands.Three ?-strands??1-?3-?4?comprised an antiparallel P-sheet.The fourth strand ?2,packs against the equivalent sheet??1'-?3'-?4'?of a symmetry related molecule to form a dimer comprising an 8 stranded P-sandwich structure where each monomer has the topological arrangement ?A-??-?1-??2'?-?3-(34.The homodimer interface stabilized by hydrogen bonds and salt bridges.Secondary structure alignment and structure overlay analysis was carried out,and MGG₀1005 were identified as belonging to the Tctexl family.Compared to D.melanogaster,S.cerevisiae and mammals,the sequence of MoTctex1 was less conservative.Three amino acid residues regions 59-65,80-112 and 138-143 in MoTctex1 were longer than those in other species.And the surface charge of a protein was also different from that of other species.It was found that MoDIC2 interacted with MoTctexl in M.oryzae by pull-down and yeast two-hybridization.To determine which region was responsible for the interaction,a deletion series of MoDIC2 was analyzed,and finally only MoDIC122-141 was found to be responsible for the interaction with MoTctexl.The mixture of MoTctexl and MoDIC117-150 was co-crystallized and we obtained the complex crystal.The structure of MoTctex1-MoDyn1I2117-151 complex were determined.Together with the data of yeast two hybrid analysis,we confirmed that MoDIC122-141 interacted with N-terminus of MoTctexl and the region from residues 80-112 participated in the interaction.It shares that two dimer of MoTctexl bind to one peptide in a asymmetric unit.The interactions in the second interface between the peptide and MoTctexl should be formed by crystal packing by ITC and Y2H analysis.The interaction analysis and mutagenesis studies reveals that the residues H34,H115 and N46,are key residues on IC binding and the EXPP motif of MoDIC2 is a necessary area.