Crystal structure of the crenarchaeal ExoIII AP endonuclease SisExoIII reveals a conserved disulfide bond endowing the protein with thermostability
Crystal structure of the crenarchaeal ExoIII AP endonuclease SisExoIII reveals a conserved disulfide bond endowing the protein with thermostability
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颅缝 ExoIII AP 核酸内切酶 SisExoIII 的晶体结构揭示了保守的二硫键，赋予蛋白质热稳定性
DOI：
10.1016/j.bbrc.2017.06.116
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发表时间：
2017
影响因子：
3.1
通讯作者：
Shen Yulong
中科院分区：
文献类型：
--
作者：
Yan Zhou;Yuan Zenglin;Ni Jinfeng;Gu Lichuan;Shen Yulong
AP 核酸内切酶识别并切割脱嘌呤/脱嘧啶 (AP) 位点，并在碱基切除修复中发挥关键作用。许多 ExoIII 和 EndoIV 家族 AP 核酸内切酶已在真核生物和细菌中进行了生化和结构表征。然而，对广古菌的研究相对较少，并且没有关于来自泉古菌的 AP 核酸内切酶的报道。在这里，我们首次报道了来自SulfolobusislandicusREY15A的颅缝ExoIII AP核酸内切酶SisExoIII的晶体结构。 SisExoIII包含由10个β-折叠形成的两层核心和由9个围绕的α-螺旋形成的壳。连接β8和β9的二硫键由Cys142和Cys215形成。这种分子内连接在天穹ExoIII同源物中是保守的，定点诱变表明它赋予蛋白质热稳定性，然而，二硫键的破坏对AP核酸内切酶活性仅产生轻微影响。我们还观察到催化中心内的几个关键残基（包括保守的 Glu35 和 Asn9）与已知的 ExoIII 蛋白相比表现出不同的构象，并形成各种分子内盐桥。该蛋白质拥有三个假定的 DNA 结合环，与其他生物体的 ExoIII 相比，具有更高的灵活性和疏水性。这些特征可能导致SisExoIII的AP核酸内切酶活性低和核酸外切酶活性缺陷。该研究加深了我们对穹窿ExoIII催化结构基础的理解，并阐明了二硫键在维持蛋白质热稳定性中的作用。
AP endonuclease recognizes and cleaves apurinic/apyrimidinic (AP) sites and plays a critical role in base excision repair. Many ExoIII and EndoIV family AP endonucleases have been characterized both biochemically and structurally in Eukaryote and Bacteria. However, relatively fewer have been studied in Euryarchaeota and there is no such report on an AP endonuclease from Crenarchaeota. Here we report, for the first time, the crystal structure of a crenarchaeal ExoIII AP endonuclease, SisExoIII, fromSulfolobusislandicusREY15A. SisExoIII comprises a two-layer core formed by 10 β-sheets and a shell formed by 9 surrounding α-helices. A disulfide bond connecting β8 and β9 is formed by Cys142 and Cys215. This intra-molecular linkage is conserved among crenarchaeal ExoIII homologs and site-directed mutagenesis revealed that it endows the protein with thermostability, however, disruption of the disulfide bond only has a slight effect on the AP endonuclease activity. We also observed that several key residues within the catalytic center including conserved Glu35 and Asn9 show different conformation compared with known ExoIII proteins and form various intra-molecular salt bridges. The protein possesses three putative DNA binding loops with higher flexibility and hydrophobicity than those of ExoIIIs from other organisms. These features may result in low AP endonuclease activity and defect of exonuclease activity of SisExoIII. The study has deepened our understanding in the structural basis of crenarchaeal ExoIII catalysis and clarified a role of the disulfide bond in maintaining protein thermostability.