Analysis of high-affinity binding of protein kinase R to double-stranded RNA.
Analysis of high-affinity binding of protein kinase R to double-stranded RNA.
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DOI：
10.1021/bi301226h
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发表时间：
2012-11-06
期刊：
影响因子：
2.9
通讯作者：
Cole JL
中科院分区：
文献类型：
--
作者：
Husain B;Mukerji I;Cole JL
蛋白激酶R(PKR)是一种干扰素诱导的蛋白激酶，在病毒感染的先天免疫应答中起着关键作用。PKR在与dsRNA结合时被激活。我们以前对PKR与dsRNA结合的分析支持一种二聚化激活模型，其中30bp代表结合两个PKR单体并通过自动磷酸化激活PKR所需的最小长度。这些研究因蛋白质-RNA聚集体的形成而变得复杂，特别是在使用较长dsRNA的低盐浓度下。在这里，我们利用荧光检测的分析性超速离心法提供的增强的灵敏度，将RNA浓度从微摩尔降低到纳摩尔。在这些条件下，我们能够在75 mM的氯化钠中表征PKR与较长的dsRNA的高亲和力结合。在较低的盐分下，PKR结合的化学计量比增加，但仍然低于以前获得的dsRNA结合域。极限PKR结合化学计量对dsRNA长度的依赖不符合非特异性结合的标准模型，表明与更长序列的结合是通过一种不同的结合模式进行的，具有更大的位点大小。虽然二聚作用在PKR的激活机制中起着关键作用，但较短的dsRNA与两个PKR单体结合的能力不足以诱导自磷酸化。我们认为，较长的RNA激活PKR与另一种结合模式有关，在这种模式下，两个dsRNA结合基序都与RNA接触，通过激酶域的直接相互作用诱导PKR二聚化。
Protein kinase R (PKR) is an interferon-induced kinase which plays a pivotal role in the innate immunity response to viral infection. PKR is activated upon binding to dsRNA. Our previous analysis of PKR binding to dsRNAs ranging from 20-40 bp supports a dimerization model for activation where 30 bp represents the minimal length required to bind two PKR monomers and activate PKR via autophosphorylation. These studies were complicated by the formation of protein-RNA aggregates, particularly at low salt concentrations using longer dsRNAs. Here, we have taken advantage of the enhanced sensitivity afforded using fluorescence-detected analytical ultracentrifugation to reduce the RNA concentrations from micromolar to nanomolar. Under these conditions, we are able to characterize high affinity binding of PKR to longer dsRNAs in 75 mM NaCl. The PKR binding stoichiometries are increased at lower salt but remain lower than those previously obtained for the dsRNA binding domain. The dependence of the limiting PKR binding stoichiometries on dsRNA length does not conform to standard models for nonspecific binding and suggests that binding to longer sequences occurs via a different binding mode with a larger site size. Although dimerization plays a key role in the PKR activation mechanism, the ability of shorter dsRNAs to bind two PKR monomers is not sufficient to induce autophosphorylation. We propose that activation of PKR by longer RNAs is correlated with an alternative binding mode where both of the dsRNA binding motifs contact the RNA, inducing PKR to dimerize via a direct interaction of the kinase domains.
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