Analysis of crystal structure of Arabidopsis MPK6 and generation of its mutants with higher activity.
Analysis of crystal structure of Arabidopsis MPK6 and generation of its mutants with higher activity.
复制标题
拟南芥MPK6晶体结构分析及高活性突变体的产生
DOI：
10.1038/srep25646
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发表时间：
2016-05-10
影响因子：
4.6
通讯作者：
Ren D
中科院分区：
文献类型：
--
作者：
Wang B;Qin X;Wu J;Deng H;Li Y;Yang H;Chen Z;Liu G;Ren D
丝裂原活化蛋白激酶（Mitogen-activated protein kinase，MAPK）级联是真核生物中高度保守的信号转导模块，在调节生长、发育和应激反应中发挥重要作用。酵母和动物MAPKs的结构已被解析，并产生了基于结构的突变体用于功能分析，但植物MAPKs的结构仍未被解析。在这里，我们报告的晶体结构的拟南芥MPK 6在3.0纳米分辨率。虽然MPK 6与ERK 2和p38在拓扑结构上相似，但MPK 6中的富含甘氨酸的环、MAPK插入物、底物结合位点和L16环的结构与ERK 2和p38的结构显示出显著差异。在结构比较的基础上，我们构建了MPK 6突变体，并分析了其在体外和植物中的激酶活性。MPK 6 F364 L和MPK 6 F368 L突变体，其中L16环中的Phe 364和Phe 368分别改变为Leu，获得了更高的内在激酶活性，并保留了正常的MAPKK激活特性。具有基础活性的MPK 6突变体的表达足以诱导Camalexin生物合成;然而，为了诱导乙烯和叶片衰老，需要具有更高活性的MPK 6突变体的表达。结果表明，这些突变体可以用来分析MPK 6的特定生物学功能。
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. MPK6F364L and MPK6F368L 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.