Crystal structure of the human CNOT6L nuclease domain reveals strict poly(A) substrate specificity
Crystal structure of the human CNOT6L nuclease domain reveals strict poly(A) substrate specificity
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人 CNOT6L 核酸酶结构域的晶体结构揭示了严格的 Poly(A) 底物特异性
DOI：
10.1038/emboj.2010.152
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发表时间：
2010-08-04
期刊：
影响因子：
11.4
通讯作者：
Rao, Zihe
中科院分区：
文献类型：
--
作者：
Wang, Hui;Morita, Masahiro;Rao, Zihe
CCR4是一种进化上保守的CCR4- not复合体成员，是主要的细胞质deadenylase。它含有一个与内切酶-外切酶-磷酸酶（EEP）家族酶同源的c端核酸酶结构域。我们利用单波长异常色散方法，用x射线晶体学测定了CCR4的人类同源物CNOT6L的核酸酶结构域的高分辨率三维结构。EEP家族的deadenylase的第一个结构采用与APE1相似的高度保守活性位点残基的水解酶典型的完整的α / β三明治折叠。CNOT6L的活性位点应该通过其带负电荷的表面识别RNA底物。体外死烯酰化酶实验证实了关键活性位点残基，并表明CNOT6L的核酸酶结构域具有完全的Mg2+依赖性死烯酰化酶活性，具有严格的poly(A) RNA底物特异性。为了了解poly(A) RNA底物结合的结构基础，我们还测定了与AMP和poly(A) DNA复合物中CNOT6L核酸酶结构域的晶体结构。由此产生的结构表明，涉及五价磷酸盐转变的分子deadenylase机制。EMBO杂志（2010）29,2566-2576。doi: 10.1038 / emboj.2010.152;2010年7月13日在线发布
CCR4, an evolutionarily conserved member of the CCR4-NOT complex, is the main cytoplasmic deadenylase. It contains a C-terminal nuclease domain with homology to the endonuclease-exonuclease-phosphatase (EEP) family of enzymes. We have determined the high-resolution three-dimensional structure of the nuclease domain of CNOT6L, a human homologue of CCR4, by X-ray crystallography using the single-wavelength anomalous dispersion method. This first structure of a deadenylase belonging to the EEP family adopts a complete alpha/beta sandwich fold typical of hydrolases with highly conserved active site residues similar to APE1. The active site of CNOT6L should recognize the RNA substrate through its negatively charged surface. In vitro deadenylase assays confirm the critical active site residues and show that the nuclease domain of CNOT6L exhibits full Mg2+-dependent deadenylase activity with strict poly(A) RNA substrate specificity. To understand the structural basis for poly(A) RNA substrate binding, crystal structures of the CNOT6L nuclease domain have also been determined in complex with AMP and poly(A) DNA. The resulting structures suggest a molecular deadenylase mechanism involving a pentacovalent phosphate transition. The EMBO Journal (2010) 29, 2566-2576. doi:10.1038/emboj.2010.152; Published online 13 July 2010