Structural and functional analyses of the severe acute respiratory syndrome coronavirus endoribonuclease Nsp15.
Structural and functional analyses of the severe acute respiratory syndrome coronavirus endoribonuclease Nsp15.
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DOI：
10.1074/jbc.m708375200
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发表时间：
2008-02-08
期刊：
影响因子：
--
通讯作者：
Kao CC
中科院分区：
文献类型：
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作者：
Bhardwaj K;Palaninathan S;Alcantara JMO;Li Yi L;Guarino L;Sacchettini JC;Kao CC
严重急性呼吸综合征（SARS）冠状病毒编码几种RNA加工酶，这些酶在RNA病毒中是不常见的，包括Nsp 15（非结构蛋白15），一种优先切割尿苷3′的六聚体核糖核酸内切酶。我们解决了Nsp15的催化失活突变体版本的结构，它被结晶为六聚体。该结构在每个亚基的活性位点中包含未报道的灵活性。活性位点残基丝氨酸293和脯氨酸343的取代允许Nsp 15在胞苷酸处裂解，而亮氨酸345的突变使得Nsp 15能够在嘌呤和嘧啶处裂解。靶向参与亚基相互作用的残基的突变通常导致形成无催化活性的单体。通过可逆交联、RNA亲和纯化和肽指纹图谱相结合的方法对RNA结合残基进行定位。Nsp 15的RNA接触部分中的几个残基的丙氨酸取代不影响六聚体的形成，但降低了RNA结合的亲和力并降低了核酸内切酶活性。这表明Nsp15六聚体与RNA相互作用的模型。
The severe acute respiratory syndrome (SARS) coronavirus encodes several RNA-processing enzymes that are unusual for RNA viruses, including Nsp15 (nonstructural protein 15), a hexameric endoribonuclease that preferentially cleaves 3′ of uridines. We solved the structure of a catalytically inactive mutant version of Nsp15, which was crystallized as a hexamer. The structure contains unreported flexibility in the active site of each subunit. Substitutions in the active site residues serine 293 and proline 343 allowed Nsp15 to cleave at cytidylate, whereas mutation of leucine 345 rendered Nsp15 able to cleave at purines as well as pyrimidines. Mutations that targeted the residues involved in subunit interactions generally resulted in the formation of catalytically inactive monomers. The RNA-binding residues were mapped by a method linking reversible cross-linking, RNA affinity purification, and peptide fingerprinting. Alanine substitution of several residues in the RNA-contacting portion of Nsp15 did not affect hexamer formation but decreased the affinity of RNA binding and reduced endonuclease activity. This suggests a model for Nsp15 hexamer interaction with RNA.