The structure of the zinc finger domain from human splicing factor ZNF265 fold
The structure of the zinc finger domain from human splicing factor ZNF265 fold
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DOI：
10.1074/jbc.m301896200
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发表时间：
2003-06-20
影响因子：
4.8
通讯作者：
Mackay, JP
中科院分区：
文献类型：
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作者：
Plambeck, CA;Kwan, AHY;Mackay, JP
负责RNA识别的蛋白质结构域的鉴定已经落后于蛋白质- DNA相互作用的表征。然而，现在越来越清楚的是，一系列的结构基序结合到RNA和它们的结构和分子作用机制开始被阐明。在本报告中，我们表达并纯化了来自ZNF 265的两个推定的RNA结合结构域之一，ZNF 265是一种已显示与剪接体组分U1- 70 K和U2 AF结合并指导选择性剪接的蛋白质（35）。我们表明，这个域，其中包含四个高度保守的半胱氨酸残基，形成一个稳定的，单体结构后，添加1摩尔当量的锌（II）。对该结构域溶液结构的测定揭示了包含两个堆叠的β-发夹结构的构象，这两个β-发夹结构的取向彼此接近80度，并包裹着锌离子;折叠类似于锌结合结构域的锌带类，尽管比该类的大多数成员少一条β-链。结构分析揭示了一个惊人的相似之处，已知的RNA结合基序的关键表面残基的分布负责使RNA接触，尽管完全缺乏结构同源性。此外，我们使用RNA凝胶迁移试验证明ZNF 265的单个交叉指结构域能够结合RNA信息。总之，这些结果定义了一个新的RNA结合基序，并应提供洞察的功能，> 100个未表征的蛋白质的序列数据库中含有这个域。
Identification of the protein domains that are responsible for RNA recognition has lagged behind the characterization of protein- DNA interactions. However, it is now becoming clear that a range of structural motifs bind to RNA and their structures and molecular mechanisms of action are beginning to be elucidated. In this report, we have expressed and purified one of the two putative RNA- binding domains from ZNF265, a protein that has been shown to bind to the spliceosomal components U1- 70K and U2AF(35) and to direct alternative splicing. We show that this domain, which contains four highly conserved cysteine residues, forms a stable, monomeric structure upon the addition of 1 molar eq of Zn( II). Determination of the solution structure of this domain reveals a conformation comprising two stacked beta- hairpins oriented at similar to80degrees to each other and sandwiching the zinc ion; the fold resembles the zinc ribbon class of zinc- binding domains, although with one less beta- strand than most members of the class. Analysis of the structure reveals a striking resemblance to known RNA-binding motifs in terms of the distribution of key surface residues responsible for making RNA contacts, despite a complete lack of structural homology. Furthermore, we have used an RNA gel shift assay to demonstrate that a single crossed finger domain from ZNF265 is capable of binding to an RNA message. Taken together, these results define a new RNA- binding motif and should provide insight into the functions of the > 100 uncharacterized proteins in the sequence data bases that contain this domain.