Two distinct nucleic acid binding surfaces of Cdc5 regulate development.
Two distinct nucleic acid binding surfaces of Cdc5 regulate development.
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DOI：
10.1042/bcj20190502
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发表时间：
2019-11
期刊：
影响因子：
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通讯作者：
Chao Wang;Mu Li;Guorui Li;Xinsen Liu;Wensheng Zhao;Bin Yu;Junfeng Liu;Jun Yang;You-Liang Peng
中科院分区：
文献类型：
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作者：
Chao Wang;Mu Li;Guorui Li;Xinsen Liu;Wensheng Zhao;Bin Yu;Junfeng Liu;Jun Yang;You-Liang Peng
细胞分裂周期 5 (Cdc5) 是真核生物中高度保守的核酸结合蛋白，在发育中发挥着关键作用。 Cdc5可以通过其N端DNA结合域(DBD)同时结合DNA和RNA，但描述其核酸识别和通过其核酸结合调节发育的分子机制仍不清楚。在此，我们展示了来自稻瘟病菌 Magnaporthe oryzae 的 MoCdc5 (MoCdc5-DBD) N 端 DNA 结合结构域的晶体结构。 MoCdc5的残基K100位于由K42、K45、R47和N92形成的带正电荷的凹槽的外围，并且在进化上是保守的。 K100 的突变显着降低了 MoCdc5-DBD 与 Cdc5 结合元件的亲和力，但不降低与常规成髓细胞增多症 (Myb) 结构域结合元件的亲和力，表明 K100 是与 Cdc5 结合元件高结合亲和力的关键残基。另一个保守残基（R31）位于剪接体结构中靠近U6 RNA的位置，其突变显着降低了MoCdc5-DBD对U6 RNA的结合能力。重要的是，这些关键残基（包括 MoCdc5 的拟南芥直系同源物 AtCDC5 中的 R31、K42 和 K100）的突变极大地损害了 AtCDC5 的功能，导致多效性发育缺陷和初级 microRNA 转录物水平降低。综上所述，我们的研究结果表明，Cdc5-DBD 通过两个不同的结合表面结合核酸，一个用于 DNA，另一个用于 RNA，这共同有助于通过核酸结合建立 Cdc5 对发育的调节机制。
Cell division cycle 5 (Cdc5) is a highly conserved nucleic acid-binding protein among eukaryotes and plays critical roles in development. Cdc5 can simultaneously bind to DNA and RNA by its N-terminal DNA-binding domain (DBD), but molecular mechanisms describing its nucleic acid recognition and the regulation of development through its nucleic acid binding remain unclear. Herein, we present a crystal structure of the N-terminal DNA-binding domain of MoCdc5 (MoCdc5-DBD) from the rice blast fungus Magnaporthe oryzae. Residue K100 of MoCdc5 is on the periphery of a positively charged groove that is formed by K42, K45, R47, and N92, and is evolutionally conserved. Mutation of K100 significantly reduces the affinity of MoCdc5-DBD to a Cdc5-binding element but not to a conventional myeloblastosis (Myb) domain-binding element, suggesting that K100 is a key residue of the high binding affinity to Cdc5-binding element. Another conserved residue (R31) is located close to the U6 RNA in the structure of the spliceosome, and its mutation dramatically reduces the binding capacity of MoCdc5-DBD for U6 RNA. Importantly, mutations in these key residues, including R31, K42, and K100 in AtCDC5, an Arabidopsis thaliana ortholog of MoCdc5, greatly impair the functions of AtCDC5, resulting in pleiotropic development defects and reduced levels of primary microRNA transcripts. Taken together, our findings suggest that Cdc5-DBD binds nucleic acids with two distinct binding surfaces, one for DNA and another for RNA, which together contribute to establishing the regulation mechanism of Cdc5 on development through nucleic acid binding.