Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of Life Sciences, Central China Normal University, Wuhan, China.
Structural Genomics Consortium, University of Toronto, Canada.
FEBS J. 2019 Aug;286(16):3240-3254. doi: 10.1111/febs.14850. Epub 2019 May 2.
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.
MBD3 是一种含有甲基-CpG 结合域（MBD）的蛋白质，是 Mi-2/NuRD 复合物的核心亚基。最近的报道表明，MBD3 可以识别甲基化 CG（mCG）和羟甲基化 CG（hmCG）的 DNA，并且对 hmCG 有偏好。然而，MBD3-MBD 是否确实具有甲基-CG 结合能力存在争议。在这项研究中，我们提供了结构基础来支持 MBD3-MBD 结合含 mCG 的 DNA 的能力。我们发现，MBD3-MBD 通过两个保守的精氨酸手指与含 mCG 的 DNA 结合，并优先结合 mCG 而不是 hmCG，与其他甲基-CpG 结合的 MBD 蛋白相似。与最接近的同源物 MBD2 相比，MBD3 中 Phe34 处的酪氨酸到苯丙氨酸取代导致其与 mCG DNA 的结合能力较弱。基于 MBD3-MBD 与非回文 AmCGC DNA 的复合物结构，我们推测所有的 mCG 结合 MBD 结构域都可以识别无取向选择性的含 mCG 的 DNA，这与我们的观察结果一致，即 mCG 二核苷酸以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保守的，大多数无脊椎动物只有一个 MBD 基因，MBD2/3。我们还研究了一些无脊椎动物 MBD2/3 的 mCG DNA 结合能力，发现保守的精氨酸手指和保守的结构折叠是无脊椎动物 MBD2/3-MBD 结合甲基化 DNA 所必需的。因此，我们的结果表明，嵌入保守结构折叠的 mCG 结合精氨酸手指是 MBD2/3 结合后生动物甲基化 DNA 的必需结构特征。
