Tianjin Key Laboratory of Protein Science, College of Life Sciences, Nankai University, Tianjin, PR China.
EMBO J. 2010 Aug 4;29(15):2566-76. doi: 10.1038/emboj.2010.152. Epub 2010 Jul 13.
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 Mg(2+)-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.
CCR4 是 CCR4-NOT 复合物中进化上保守的成员，是主要的细胞质脱腺苷酶。它包含一个 C 末端核酸酶结构域，与内切核酸酶-外切核酸酶-磷酸酶 (EEP) 家族的酶具有同源性。我们通过 X 射线晶体学使用单波长异常分散法确定了 CNOT6L（CCR4 的人类同源物）的核酸酶结构域的高分辨率三维结构。属于 EEP 家族的第一个脱腺苷酶结构采用完整的α/β三明治折叠，这是水解酶的典型特征，具有高度保守的活性位点残基，类似于 APE1。CNOT6L 的活性位点应通过其带负电荷的表面来识别 RNA 底物。体外脱腺苷酶测定证实了关键的活性位点残基，并表明 CNOT6L 的核酸酶结构域具有完整的 Mg2+依赖性脱腺苷酶活性，并具有严格的多聚（A）RNA 底物特异性。为了了解多聚（A）RNA 底物结合的结构基础，还确定了 CNOT6L 核酸酶结构域与 AMP 和多聚（A）DNA 复合物的晶体结构。所得结构表明涉及五价磷酸过渡的分子脱腺苷酶机制。
