Identification of intersubunit domain interactions within eukaryotic initiation factor (eIF) 2B, the nucleotide exchange factor for translation initiation.
Identification of intersubunit domain interactions within eukaryotic initiation factor (eIF) 2B, the nucleotide exchange factor for translation initiation.
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DOI：
10.1074/jbc.m111.331645
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发表时间：
2012-03-09
期刊：
影响因子：
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通讯作者：
Pavitt GD
中科院分区：
文献类型：
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作者：
Reid PJ;Mohammad-Qureshi SS;Pavitt GD
背景：eIF 2B是一种重要的翻译因子和蛋白质合成调节因子，与人类疾病有关。结果：鉴定了五亚基eIF 2B复合物内的蛋白质-蛋白质相互作用。结论：eIF 2B复合物的形成需要广泛的亚基结构域间的相互作用。意义：第一个实验模型提出了eIF 2B γ和β亚基相互作用。在真核生物翻译起始中，eIF 2B是G蛋白eIF 2在蛋白质合成起始之间重新激活所需的鸟嘌呤核苷酸交换因子（GEF）。eIF 2B异常复杂，具有GEF活性所必需的5个亚基（α-κ B），并通过eIF 2 α的磷酸化进行控制。此外，eIF 2B中的遗传性突变会导致致命的白质脑病。在这里，我们描述了实验检查eIF 2B γ和eIF 2B γ的结构域，它们都与磷酸己糖核苷酸焦磷酸化酶家族共享序列和预测的三级结构相似性。首先，使用遗传学方法，我们没有发现任何证据来支持一个潜在的核苷酸结合区域内的焦磷酸化酶样结构域（PLD）的eIF 2B的核苷酸交换的显着作用。这些发现与先前提出的核苷酸交换机制不一致。通过使用一系列构建体和共表达和沉淀策略，我们发现eIF 2B的β和γ PLD以及预测形成左手β螺旋的共享第二结构域对于eIF 2B复合物形成所必需的eIF 2B亚基之间的蛋白间相互作用都是至关重要的。我们已经确定了形成eIF 2B γ-亚复合物的PLD和左手β螺旋结构域之间的广泛相互作用，并提出了eIF 2B亚基之间结构域相互作用的模型。
Background: eIF2B is a critical translation factor and regulator of protein synthesis that is implicated in human disease. Results: Protein-protein interactions within the five-subunit eIF2B complex are identified. Conclusion: eIF2B complex formation requires extensive intersubunit domain interactions. Significance: The first experimental model is proposed for eIF2Bγ and -ϵ subunit interactions. In eukaryotic translation initiation, eIF2B is the guanine nucleotide exchange factor (GEF) required for reactivation of the G protein eIF2 between rounds of protein synthesis initiation. eIF2B is unusually complex with five subunits (α–ϵ) necessary for GEF activity and its control by phosphorylation of eIF2α. In addition, inherited mutations in eIF2B cause a fatal leukoencephalopathy. Here we describe experiments examining domains of eIF2Bγ and ϵ that both share sequence and predicted tertiary structure similarity with a family of phospho-hexose sugar nucleotide pyrophosphorylases. Firstly, using a genetic approach, we find no evidence to support a significant role for a potential nucleotide-binding region within the pyrophosphorylase-like domain (PLD) of eIF2Bϵ for nucleotide exchange. These findings are at odds with one mechanism for nucleotide exchange proposed previously. By using a series of constructs and a co-expression and precipitation strategy, we find that the eIF2Bϵ and -γ PLDs and a shared second domain predicted to form a left-handed β helix are all critical for interprotein interactions between eIF2B subunits necessary for eIF2B complex formation. We have identified extensive interactions between the PLDs and left-handed β helix domains that form the eIF2Bγϵ subcomplex and propose a model for domain interactions between eIF2B subunits.