Structural analyses reveal that MBD3 is a methylated CG binder
Structural analyses reveal that MBD3 is a methylated CG binder
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结构分析表明 MBD3 是甲基化 CG 结合剂
DOI：
10.1111/febs.14850
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发表时间：
2019-08-01
期刊：
影响因子：
5.4
通讯作者：
Min, Jinrong
中科院分区：
文献类型：
--
作者：
Liu, Ke;Lei, Ming;Min, Jinrong
MBD 3是一种含有甲基-CpG结合结构域（MBD）的蛋白，是Mi-2/NuRD复合物的核心亚基。最近的报告显示，MBD 3识别甲基化CG（mCG）和羟甲基化CG（hmCG）的DNA，优先选择hmCG。然而，MBD 3-MBD是否确实具有甲基CG结合能力是有争议的。在这项研究中，我们提供了支持MBD 3-MBD结合含mCG的DNA的能力的结构基础。我们发现MBD 3-MBD通过两个保守的精氨酸指与含mCG的DNA结合，并且优先与mCG结合而不是hmCG，类似于其他甲基-CpG结合的MBD蛋白。与其最接近的同源物MBD 2相比，MBD 3的Phe 34处的酪氨酸至苯丙氨酸取代是导致较弱的mCG DNA结合能力的原因。基于MBD 3-MBD与非回文AmCGC DNA的复杂结构，我们认为所有的mCG结合MBD结构域都可以识别含有mCG的DNA，而没有方向选择性，这与我们的观察结果一致，即mCG二核苷酸外的序列不会显著影响mCG DNA结合。DNA胞嘧啶甲基化在大多数后生动物中是进化保守的，并且大多数无脊椎动物只有一个MBD基因，MBD 2/3。我们还研究了一些无脊椎动物MBD 2/3的mCG DNA结合能力，发现保守的精氨酸指和保守的结构折叠是无脊椎动物中MBD 2/3-MBD结合甲基化DNA所必需的。因此，我们的研究结果表明，嵌入到一个保守的结构折叠的mCG-结合精氨酸指MBD 2/3s之间的后生动物与甲基化DNA结合的基本结构特征。
The MBD3, a methyl-CpG-binding domain (MBD)-containing protein, is a core subunit of the Mi-2/NuRD complex. Recent reports show that MBD3 recognizes both methylated CG (mCG)- and hydroxymethylated CG (hmCG)-containing DNA, with a preference for hmCG. However, whether the MBD3-MBD indeed has methyl-CG-binding ability is controversial. In this study, we provided the structural basis to support the ability of MBD3-MBD to bind mCG-containing DNA. We found that the MBD3-MBD bound to mCG-containing DNA through two conserved arginine fingers, and preferentially bound to mCG over hmCG, similar to other methyl-CpG-binding MBD proteins. Compared to its closest homolog MBD2, the tyrosine-to-phenylalanine substitution at Phe34 of MBD3 is responsible for a weaker mCG DNA binding ability. Based on the complex structure of MBD3-MBD with a nonpalindromic AmCGC DNA, we suggest that all the mCG-binding MBD domains can recognize mCG-containing DNA without orientation selectivity, consistent with our observations that the sequences outside the mCG dinucleotide do not affect mCG DNA binding significantly. DNA cytosine methylation is evolutionarily conserved in most metazoans, and most invertebrates have only one MBD gene, MBD2/3. We also looked into the mCG DNA binding ability of some invertebrates MBD2/3 and found that the conserved arginine fingers and a conserved structural fold are required for methylated DNA binding by MBD2/3-MBDs in invertebrates. Hence, our results demonstrate that mCG-binding arginine fingers embedded into a conserved structural fold are essential structural features for MBD2/3s binding to methylated DNA among metazoans.