Nikolova Evgenia N, Stanfield Robyn L, Dyson H Jane, Wright Peter E
Department of Integrative Structural and Computational Biology and Skaggs Institute of Chemical Biology , The Scripps Research Institute , 10550 North Torrey Pines Road , La Jolla , California 92037 , United States.
Biochemistry. 2018 Apr 10;57(14):2109-2120. doi: 10.1021/acs.biochem.8b00065. Epub 2018 Mar 26.
Many eukaryotic transcription factors recognize the epigenetic marker 5-methylcytosine (mC) at CpG sites in DNA. Despite their structural diversity, methyl-CpG-binding proteins (MBPs) share a common mode of recognition of mC methyl groups that involves hydrophobic pockets and weak hydrogen bonds of the CH···O type. The zinc finger protein Kaiso possesses a remarkably high specificity for methylated over unmethylated CpG sites. A key contribution to this specificity is provided by glutamate 535 (E535), which is optimally positioned to form multiple interactions with mCpG, including direct CH···O hydrogen bonds. To examine the role of E535 and CH···O hydrogen bonding in the preferential recognition of mCpG sites, we determined the structures of wild type Kaiso (WT) and E535 mutants and characterized their interactions with methylated DNA by nuclear magnetic resonance spectroscopy (NMR), X-ray crystallography, and in vitro protein-DNA binding assays. Our data show that Kaiso favors an mCpG over a CpG site by 2 orders of magnitude in affinity and that an important component of this effect is the presence of hydrophobic and CH···O contacts involving E535. Moreover, we present the first direct evidence for formation of a CH···O hydrogen bond between an MBP and 5-methylcytosine by using experimental (NMR) and quantum mechanical chemical shift analysis of the mC methyl protons. Together, our findings uncover a critical function of methyl-specific interactions, including CH···O hydrogen bonds, that optimize the specificity and affinity of MBPs for methylated DNA and contribute to the precise control of gene expression.
许多真核转录因子可识别DNA中CpG位点的表观遗传标记5-甲基胞嘧啶（mC）。尽管甲基-CpG结合蛋白（MBP）结构多样，但它们识别mC甲基基团的方式相同，都涉及疏水口袋和CH···O型弱氢键。锌指蛋白Kaiso对甲基化的CpG位点具有极高的特异性。谷氨酸535（E535）对这种特异性起关键作用，它的位置理想，可与mCpG形成多种相互作用，包括直接的CH···O氢键。为研究E535和CH···O氢键在优先识别mCpG位点中的作用，我们测定了野生型Kaiso（WT）和E535突变体的结构，并通过核磁共振光谱（NMR）、X射线晶体学和体外蛋白质-DNA结合试验来表征它们与甲基化DNA的相互作用。我们的数据表明，Kaiso对mCpG位点的亲和力比对CpG位点高2个数量级，这种效应的一个重要组成部分是存在涉及E535的疏水和CH···O接触。此外，我们通过对mC甲基质子进行实验（NMR）和量子力学化学位移分析，首次直接证明了MBP与5-甲基胞嘧啶之间形成了CH···O氢键。总之，我们的研究结果揭示了甲基特异性相互作用的关键功能，包括CH···O氢键，这些相互作用优化了MBP对甲基化DNA的特异性和亲和力，并有助于精确控制基因表达。
