Switching Metal Ion Coordination and DNA Recognition in a Tandem CCHHC-type Zinc Finger Peptide
Switching Metal Ion Coordination and DNA Recognition in a Tandem CCHHC-type Zinc Finger Peptide
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DOI：
10.1021/ic4003516
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发表时间：
2013-04-15
影响因子：
4.6
通讯作者：
Michel, Sarah L. J.
中科院分区：
文献类型：
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作者：
Besold, Angelique N.;Oluyadi, Abdulafeez A.;Michel, Sarah L. J.
神经锌指因子-1 （Neural Zinc Finger Factor-1, NZF-1）和髓鞘转录因子1 （Myelin Transcription Factor 1, MyT1）是两个同源的非经典锌指蛋白，参与中枢神经系统（central nervous system， CNS）的发育。NZF-1和MyT1都含有多个ZF结构域，每个结构域都包含一个绝对保守的Cys(2)His(2)Cys基序。所有三种半胱氨酸和第二组氨酸已被证明配位Zn(II)；然而，第一组氨酸的作用仍未得到解决。利用含有两个ZF结构域的NZF-1的功能形式（NZF-1- f2f3），制备了每个组氨酸依次突变为苯丙氨酸的突变蛋白，以确定组氨酸残基在DNA识别中的作用。当第一个组氨酸发生突变时，该蛋白以与天然蛋白类似的方式结合Zn（11）。令人惊讶的是，该突变体不结合靶DNA (β - rar)，这表明非配位组氨酸对序列选择性DNA识别至关重要。当第二组氨酸发生突变时，第一组氨酸将配位Zn(II)；然而，蛋白质的整体折叠被扰乱导致DNA结合的废除。NZF-1-F2F3选择性结合特定的DNA靶序列（来自β - rar），具有高亲和力（nM）；而它的同系物MyT1 (MyT1- f2f3)，与NZF-1-F2F3有92%的相同，非特异性地与相同的DNA序列结合。NZF-1-F2F3中的一个非保守氨基酸残基被证明是这种高亲和力DNA与β - rar结合的原因。当这个残基（精氨酸）被改造成MyT1-F2F3序列时，对β - rar DNA的亲和力增加。
Neural Zinc Finger Factor-1 (NZF-1) and Myelin Transcription Factor 1 (MyT1) are two homologous nonclassical zinc finger (ZF) proteins that are involved in the development of the central nervous system (CNS). Both NZF-1 and MyT1 contain multiple ZF domains, each of which contains an absolutely conserved Cys(2)His(2)Cys motif. All three cysteines and the second histidine have been shown to coordinate Zn(II); however, the role of the first histidine remains unresolved. Using a functional form of NZF-1 that contains two ZF domains (NZF-1-F2F3), mutant proteins in which each histidine was sequentially mutated to a phenylalanine were prepared to determine the role(s) of the histidine residues in DNA recognition. When the first histidine is mutated, the protein binds Zn(11) in an analogous manner to the native protein. Surprisingly, this mutant does not bind to target DNA (beta-RAR), suggesting that the noncoordinating histidine is critical for sequence selective DNA recognition. The first histidine will coordinate Zn(II) when the second histidine is mutated; however, the overall fold of the protein is perturbed leading to abrogation of DNA binding. NZF-1-F2F3 selectively binds to a specific DNA target sequence (from beta-RAR) with high affinity (nM); while its homologue MyT1 (MyT1-F2F3), which is 92% identical to NZF-1-F2F3, binds to this same DNA sequence nonspecifically. A single, nonconserved amino acid residue in NZF-1-F2F3 is shown to be responsible for this high affinity DNA binding to beta-RAR. When this residue (arginine) is engineered into the MyT1-F2F3 sequence, the affinity for beta-RAR DNA increases.