title: Analysis of crystal structure of Arabidopsis MPK6 and generation of its mutants with higher activity.
abstract: Mitogen-activated protein kinase (MAPK) cascades, which are the highly conserved signalling modules in eukaryotic organisms, have been shown to play important roles in regulating growth, development, and stress responses. The structures of various MAPKs from yeast and animal have been solved, and structure-based mutants were generated for their function analyses, however, the structures of plant MAPKs remain unsolved. Here, we report the crystal structure of Arabidopsis MPK6 at a 3.0 Å resolution. Although MPK6 is topologically similar to ERK2 and p38, the structures of the glycine-rich loop, MAPK insert, substrate binding sites, and L16 loop in MPK6 show notable differences from those of ERK2 and p38. Based on the structural comparison, we constructed MPK6 mutants and analyzed their kinase activity both in vitro and in planta. MPK6(F364L) and MPK6(F368L) mutants, in which Phe364 and Phe368 in the L16 loop were changed to Leu, respectively, acquired higher intrinsic kinase activity and retained the normal MAPKK activation property. The expression of MPK6 mutants with basal activity is sufficient to induce camalexin biosynthesis; however, to induce ethylene and leaf senescence, the expression of MPK6 mutants with higher activity is required. The results suggest that these mutants can be used to analyze the specific biological functions of MPK6.
authors.affiliation: State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China.
authors.forename: Xinghua
authors.initials: X
authors.lastname: Qin
authors.affiliation: State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China.
authors.forename: Hongying
authors.initials: H
authors.lastname: Deng
authors.affiliation: State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Li
authors.affiliation: State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China.
authors.forename: Hailian
authors.initials: H
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China.
authors.forename: Zhongzhou
authors.initials: Z
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China.
authors.forename: Guoqin
authors.initials: G
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China.
authors.forename: Dongtao
authors.initials: D
authors.lastname: Ren
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018360:Crystallography, X-Ray; D005030:Ethylenes / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D007211:Indoles / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010766:Phosphorylation; D030821:Plants, Genetically Modified; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013844:Thiazoles / Q000378:metabolism; D000094162:Phytoalexins
chemical_list: D029681:Arabidopsis Proteins; D005030:Ethylenes; D007211:Indoles; D020928:Mitogen-Activated Protein Kinases; D011994:Recombinant Proteins; D013844:Thiazoles; C102405:camalexin; D000094162:Phytoalexins; C036216:ethylene; C436293:MPK6 protein, Arabidopsis