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首页> 外文期刊>Journal of Molecular Biology >Mutational Analysis of the SARS Virus Nsp15 Endoribonuclease: Identification of Residues Affecting Hexamer Formation.
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Mutational Analysis of the SARS Virus Nsp15 Endoribonuclease: Identification of Residues Affecting Hexamer Formation.

机译：SARS病毒Nsp15内切核糖核酸酶的突变分析：鉴定影响六聚体形成的残基。

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摘要

The severe acute respiratory syndrome (SARS) coronavirus virus non-structural protein 15 is a Mn(2+)-dependent endoribonuclease with specificity for cleavage at uridylate residues. To better understand structural and functional characteristics of Nsp15, 22 mutant versions of Nsp15 were produced in Escherichia coli as His-tagged proteins and purified by metal-affinity and ion-exchange chromatography. Nineteen of the mutants were soluble and were analyzed for enzymatic activity. Six mutants, including four at the putative active site, were significantly reduced in endoribonuclease activity. Two of the inactive mutants had unusual secondary structures compared to the wild-type protein, as measured by circular dichroism spectroscopy. Gel-filtration analysis, velocity sedimentation ultracentrifugation, and native gradient pore electrophoresis all showed that the wild-type protein exists in an equilibrium between hexamers and monomers in solution, with hexamers dominating at micromolar protein concentration,while native gradient pore electrophoresis also revealed the presence of trimers. A mutant in the N terminus of Nsp15 was impaired in hexamer formation and had low endoribonuclease activity, suggesting that oligomerization is required for endoribonuclease activity. This idea was supported by titration experiments showing that enzyme activity was strongly concentration-dependent, indicating that oligomeric Nsp15 is the active form. Three-dimensional reconstruction of negatively stained single particles of Nsp15 viewed by transmission electron microscopic analysis suggested that the six subunits were arranged as a dimer of trimers with a number of cavities or channels that may constitute RNA binding sites.
机译：严重急性呼吸系统综合症（SARS）冠状病毒病毒非结构蛋白15是Mn（2+）依赖性内切核糖核酸酶，具有在尿嘧啶残基处裂解的特异性。为了更好地了解Nsp15的结构和功能特性，在大肠杆菌中生产了22种Nsp15突变体形式的His标记蛋白，并通过金属亲和力和离子交换色谱法纯化。 19个突变体是可溶的，并分析了酶活性。六个突变体，包括在假定的活性位点的四个突变体，其核糖核酸内切酶活性显着降低。通过圆二色光谱法测定，与野生型蛋白相比，两个无活性的突变体具有异常的二级结构。凝胶过滤分析，速度沉降超速离心和天然梯度孔电泳均表明，野生型蛋白在溶液中六聚体和单体之间处于平衡状态，六聚体在微摩尔蛋白浓度下占主导，而天然梯度孔电泳也表明存在三聚体。 Nsp15 N末端的一个突变体的六聚体形成受到损害，并且具有较低的核糖核酸内切酶活性，这表明寡聚化是核糖核酸内切酶活性所必需的。滴定实验表明该酶活性强烈地依赖于浓度，表明寡聚Nsp15是活性形式，这一观点得到了支持。透射电子显微镜分析观察到的Nsp15负染色单个颗粒的三维重建表明，六个亚基排列为三聚体的二聚体，具有许多可能构成RNA结合位点的腔或通道。

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