Mutational analysis of the SARS virus Nsp15 endoribonuclease: identification of residues affecting hexamer formation.
Mutational analysis of the SARS virus Nsp15 endoribonuclease: identification of residues affecting hexamer formation.
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SARS病毒NSP15内切核酸酶的突变分析：影响六聚体形成的残基。
DOI：
10.1016/j.jmb.2005.09.007
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发表时间：
2005-11-11
影响因子：
5.6
通讯作者：
Kao C
中科院分区：
文献类型：
--
作者：
Guarino LA;Bhardwaj K;Dong W;Sun J;Holzenburg A;Kao C
严重急性呼吸综合征(SARS)冠状病毒非结构蛋白15是一种依赖于Mn2+的内切核酸酶，具有切割尿苷残基的特异性。为了更好地了解Nsp15的结构和功能特征，在大肠杆菌中表达了22个突变型的Nsp15作为组氨酸标记的蛋白，并通过金属亲和层析和离子交换层析进行纯化。其中19个突变体是可溶的，并对其进行了酶活性分析。6个突变体，其中4个位于假定的活性部位，内切核酸酶活性显著降低。根据圆二色谱的测量，其中两个非活性突变体与野生型蛋白相比具有不寻常的二级结构。凝胶过滤、速度沉降、超速离心法和自然梯度孔电泳法均表明野生型蛋白在溶液中以六聚体和单体之间的平衡状态存在，在微摩尔蛋白浓度范围内以六聚体为主，而自然梯度孔电泳示出三聚体的存在。Nsp15基因N端的一个突变体在六聚体形成过程中受到损害，内切核酸酶活性较低，提示内切核酸酶活性需要寡聚作用。滴定实验表明，酶的活性强烈地依赖于浓度，这表明寡聚体Nsp15是酶的活性形式。对Nsp15负染单个颗粒的三维重建表明，这六个亚基以三聚体的二聚体形式排列，其中有许多空洞或通道，这些空洞或通道可能构成RNA结合位点。
The severe acute respiratory syndrome (SARS) coronavirus virus non-structural protein 15 is a Mn2+-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.
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影响因子：
56.9
作者：
PELISKA, JA;BENKOVIC, SJ
通讯作者：
BENKOVIC, SJ
影响因子：
11.4
作者：
BAKER, SC;LAI, MMC
通讯作者：
LAI, MMC
影响因子：
5.4
作者：
SETHNA, PB;HOFMANN, MA;BRIAN, DA
通讯作者：
BRIAN, DA
影响因子：
3
作者：
Pettersen, EF;Goddard, TD;Ferrin, TE
通讯作者：
Ferrin, TE
影响因子：
2.2
作者：
HOVMOLLER, S
通讯作者：
HOVMOLLER, S