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首页> 外文期刊>Biochemistry >DNA Recognition of 5?Carboxylcytosine by a Zfp57 Mutant at an Atomic Resolution of 0.97 ?
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DNA Recognition of 5?Carboxylcytosine by a Zfp57 Mutant at an Atomic Resolution of 0.97 ?

机译：Zfp57突变体在0.97？原子分辨率下对5'羧基胞嘧啶的DNA识别。

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摘要

The Zfp57 gene encodes a KRAB (Kru?ppelassociated box) domain-containing C2H2 zinc finger transcription factor that is expressed in early development. Zfp57 protein recognizes methylated CpG dinucleotide within GCGGCA elements at multiple imprinting control regions. In the previously determined structure of the mouse Zfp57 DNA-binding domain in complex with DNA containing 5- methylcytosine (5mC), the side chains of Arg178 and Glu182 contact the methyl group via hydrophobic and van der Waals interactions. We examined the role of Glu182 in recognition of 5mC by mutagenesis. The majority of mutants examined lose selectivity of methylated (5mC) over unmodified (C) and oxidative derivatives, 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine (5caC), suggesting that the side chain of Glu182 (the size and the charge) is dispensable for methyl group recognition but negatively impacts the binding of unmodified cytosine as well as oxidized derivatives of 5mC to achieve 5mC selectivity. Substitution of Glu182 with its corresponding amide (E182Q) had no effect on methylated DNA binding but gained significant binding affinity for 5caC DNA, resulting in a binding affinity for 5caC DNA comparable to that of the wild-type protein for 5mC. We show structurally that the uncharged amide group of E182Q interacts favorably with the carboxylate group of 5caC. Furthermore, introducing a positively charged arginine at position 182 resulted in a mutant (E182R) having higher selectivity for the negatively charged 5caC.
机译：Zfp57基因编码一个包含KRAB（克鲁勃关联框）域的C2H2锌指转录因子，该因子在早期发育中表达。 Zfp57蛋白可在多个印迹控制区域识别GCGGCA元件内的甲基化CpG二核苷酸。在先前确定的小鼠Zfp57 DNA结合结构域与含有5-甲基胞嘧啶（5mC）的DNA复合的结构中，Arg178和Glu182的侧链通过疏水和范德华相互作用与甲基接触。我们检查了通过诱变Glu182在识别5mC中的作用。检查的大多数突变体都失去了甲基化（5mC）对未修饰的（C）和氧化衍生物，5-羟甲基胞嘧啶，5-甲酰基胞嘧啶和5-羧化胞嘧啶（5caC）的选择性，表明Glu182的侧链（大小和电荷）对于甲基识别是必不可少的，但会对未修饰的胞嘧啶以及5mC的氧化衍生物的结合产生负面影响，从而实现5mC的选择性。用其相应的酰胺（E182Q）取代Glu182对甲基化的DNA结合没有影响，但是获得了对5caC DNA的显着结合亲和力，从而导致了对5caC DNA的亲和力与5mC的野生型蛋白相当。我们在结构上表明，E182Q的不带电荷的酰胺基与5caC的羧酸酯基相互作用有利。此外，在182位引入带正电荷的精氨酸导致突变体（E182R）对带负电荷的5caC具有更高的选择性。

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