Extracellular Structure Analysis Of The Receptor-like Kinase TMK3 In Arabidopsis Thaliana

Transmembrane kinases(TMKs)are members of plant-like receptor protein kinases(RLKs).TMKs consist of an extracellular leucine-rich repeat(LRR)structural domain,a single transmembrane structural domain and an intracellular kinase structural domain.the LRR structural domain mainly serves as a scaffold for proteinprotein interactions and is involved in a variety of biological processes.Studies have shown that TMKs are key regulators of cell growth and cell proliferation.Despite significant progress in understanding the functions of TMKs,the ligand binding mode of TMKs is still poorly understood.In this study,in order to verify the biological functions of TMKs,we took structural biology and other related methods to obtain the three-dimensional structures of the proteins in order to try to elucidate the mechanism at the atomic level.The The main findings are as follows.(1)Vector construction.The amino acid sequence of TMK3 protein was predicted by bioinformatics software to ensure the stability of the protein and the amplified fragment was cloned into E.coli expression vector p Fast Bac-HEM to obtain the recombinant vector.(2)Insect expression system construction.The recombinant baculovirus(ba cmid)was obtained by transferring the successfully constructed p Fast Bac-HEMTMK3-ECD vector into DH10 Bac receptor state.High concentration of bacmid DNA was extracted to transfect insect cells(3)Protein expression.The successfully transfected insect cells were.subcultured to obtain P3 generation with high virus titer.The P3 generation was infected with 200 ml of insect cells and cultured at 27? in a 110r/min shaker.(4)Protein purification.We used affinity chromatography to obtain the protein purity suitable for crystallization.(5)Protein crystallization screening.Determining Kit 2-13(0.2M ammonium sulfate 0.1M sodium acetate trihydrate p H4.6 30% W/V PEG monomethyl ether 2000)as the conditions for the crystals to continue optimization.Finally,crystals suitable for diffraction were obtained by Additive reagent.(6)Protein structure resolution.After collecting the diffraction data,the crystal structure of TMK3 extracellular domain(TMK3-ECD)was determined to a resolution of 2.06 ? with Rwork of 17.69% and Rfree of 20.58%.(7)Functional speculation of TMK3 protein.Through structural analysis,we found that the extracellular structural domain of TMK3 is rich in leucine repeats(LRRs),TMK3-LRR contains 13 LRRs,and a non-LRR region exists between the 10 th and 11 th LRRs(316-364 AA).Based on the analysis,we hypothesized that non-LRR has an important role in structural integrity and ligand binding.In summary,the results of this study indicate that there is a non-LRR region with an electronegative groove-like pocket structure in the LRRs of TMK3-LRR.The non-LRR plays a critical role in structural integrity and may contribute to ligand interactions.And also this structural domain contains two pairs of disulfide bonds.By comparing the analysis with the reported RLK-LRRs containing non-LRRs,we speculate that the non-LRR structural domain has a very important biological significance.