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首页> 外文期刊>Biochemistry >Assembly Pathway of an AAA+ Protein: Tracking ClpA and ClpAP Complex Formation in Real Time
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Assembly Pathway of an AAA+ Protein: Tracking ClpA and ClpAP Complex Formation in Real Time

机译：AAA +蛋白的组装途径：实时跟踪ClpA和ClpAP复合物的形成

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The ClpAP chaperone-protease complex is active as a cylindrically shaped oligomeric complex built of the proteolytic ClpP double ring as the core of the complex and two ClpA hexamers associating with the ends of the core cylinder. The ClpA chaperone belongs to the larger family of AAA~+ ATPases and is responsible for preparing protein substrates for degradation by ClpP. Here, we study in real time using fluorescence and light scattering stopped-flow methods the complete assembly pathway of this bacterial chaperone-protease complex consisting of ATP-induced ClpA hexamer formation and the subsequent association of ClpA hexamers with the ClpP core cylinder. We provide evidence that ClpA assembles into hexamers via a tetrameric intermediate and that hexamerization coincides with the appearance of ATPase activity. While ATP-induced oligomerization of ClpA is a prerequisite for binding of ClpA to ClpP, the kinetics of ClpA hexamer formation are not influenced by the presence of ClpP. Models for ClpA hexamerization and ClpA-ClpP association are presented along with rate parameters obtained from numerical fitting procedures. The hexamerization kinetics show that the tetrameric intermediate transiently accumulates, forming rapidly at early time points and then decaying at a slower rate to generate the hexamer. The association of assembled ClpA hexamers with the ClpP core cylinder displays cooperativity, supporting the coexistence of interchanging ClpP conformations with different affinities for ClpA.
机译：ClpAP伴侣蛋白复合物具有圆柱状的寡聚复合物的活性，该复合物由蛋白水解的ClpP双环作为复合物的核心，两个ClpA六聚体与核心圆柱的末端缔合。 ClpA分子伴侣属于较大的AAA〜+ ATPase家族，负责为ClpP降解准备蛋白质底物。在这里，我们使用荧光和光散射停止流方法实时研究了该细菌伴侣蛋白复合物的完整组装路径，该复合物由ATP诱导的ClpA六聚体形成以及随后的ClpA六聚体与ClpP核心圆柱体组成。我们提供的证据表明ClpA通过四聚体中间体组装成六聚体，并且六聚化与ATPase活性的出现相吻合。尽管ATP诱导的ClpA寡聚是ClpA与ClpP结合的先决条件，但ClpA六聚体形成的动力学不受ClpP的存在的影响。介绍了ClpA六聚化和ClpA-ClpP关联的模型，以及从数值拟合程序获得的速率参数。六聚反应动力学表明，四聚体中间体会暂时积累，在早期时间点迅速形成，然后以较慢的速率分解生成六聚体。组装的ClpA六聚体与ClpP核心圆柱体的结合显示了协同作用，支持了具有不同亲和力的ClpP构象互换的ClpP构象的共存。

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