Essential role of the N-terminal domain in the regulation of RIG-I ATPase activity
Essential role of the N-terminal domain in the regulation of RIG-I ATPase activity
复制标题
DOI：
10.1074/jbc.m706777200
复制
发表时间：
2008-04-04
影响因子：
4.8
通讯作者：
Deval, Jerome
中科院分区：
文献类型：
--
作者：
Gee, Peter;Chua, Pong Kian;Deval, Jerome
维甲酸诱导基因I(RIG-I)是一种胞质受体，识别病毒RNA并激活干扰素介导的先天抗病毒反应。为了在受体水平上了解信号激活的机制，我们克隆、表达和纯化了含有两个caspase激活和募集结构域(CADS)的人RIG-I，紧随其后的是C末端解旋酶结构域。我们发现，重组RIG-I是一种与双链RNA相互作用的功能性蛋白质，与单链RNA结构相比，除非它们含有5‘-三磷酸基团，否则亲和力要高得多。病毒RNA与RIG-I结合可刺激33倍的ATP水解速度，这在细胞水平上转化为43倍的β-干扰素表达。相反，分离的ATPase/解旋酶结构域在保持其RNA配体结合特性的同时被结构性激活。这些结果支持最近的模型，根据该模型，在没有RNA的情况下，RIG-I信号通过CARD和C末端之间的分子内相互作用而被自动抑制。基于pH谱和金属离子依赖性实验，我们认为在无RNA抑制构象下，RIG-I的活性中心不能有效地容纳二价阳离子。总体而言，这些结果表明RNA结合与导致信号转导的ATPase酶功能之间存在直接关联，并表明在没有病毒RNA的情况下，CARDS对ATPase活性的严格控制阻止了RIG-I信号传递。
Retinoic acid-inducible gene I (RIG-I) is a cytosolic receptor that recognizes viral RNA and activates the interferon-mediated innate antiviral response. To understand the mechanism of signal activation at the receptor level, we cloned, expressed, and purified human RIG-I containing the two caspase activation and recruitment domains (CARDs) followed by the C-terminal helicase domain. We found that recombinant RIG-I is a functional protein that interacts with double-stranded RNA with substantially higher affinity as compared with single-stranded RNA structures unless they contain a 5'-triphosphate group. Viral RNA binding to RIG-I stimulates the velocity of ATP hydrolysis by 33-fold, which at the cellular level translates into a 43-fold increase of interferon-beta expression. In contrast, the isolated ATPase/helicase domain is constitutively activated while also retaining its RNA ligand binding properties. These results support the recent model by which RIG-I signaling is autoinhibited in the absence of RNA by intra-molecular interactions between the CARDs and the C terminus. Based on pH profile and metal ion dependence experiments, we propose that the active site of RIG-I cannot efficiently accommodate divalent cations under the RNA-free repressed conformation. Overall, these results show a direct correlation between RNA binding and ATPase enzymatic function leading to signal transduction and suggest that a tight control of ATPase activity by the CARDs prevents RIG-I signaling in the absence of viral RNA.