Functional signal peptides bind a soluble n-terminal fragment of SecA and inhibit its ATPase activity
Functional signal peptides bind a soluble n-terminal fragment of SecA and inhibit its ATPase activity
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DOI：
10.1074/jbc.m100098200
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发表时间：
2001-06-01
影响因子：
4.8
通讯作者：
Gierasch, LM
中科院分区：
文献类型：
--
作者：
Triplett, TL;Sgrignoli, AR;Gierasch, LM
SecA对前分泌蛋白的选择性识别对于大肠杆菌输出蛋白质的过程至关重要，但对于识别的要求以及前体与SecA的结合方式知之甚少，其主要原因是缺乏适合SecA-前体复合物生物物理研究的可溶性系统。使这种系统的开发变得复杂的是，只有当 SecA 通过与膜和 SecYEG 结合而被激活时，它才可能以高亲和力、高效的方式与前体相互作用。前体/SecA 相互作用的一个关键方面是它受到各种 SecA 配体（核苷酸、脂质、SecYEG）的调节，以促进前体的释放，很可能以逐步的方式进行易位。最近的一些报告表明，可以使用分离的结构域来研究 SecA 的功能。使用这种方法，我们分离了 SecA 的蛋白水解生成的 N 端片段，该片段稳定折叠，具有高 ATPase 活性，代表 SecA 的激活版本。我们在此报告，该片段（称为 SecA64）与信号肽的结合亲和力明显高于 SecA，此外，SecA64 的 ATPase 活性被信号肽抑制到一定程度，该程度与这些信号肽抑制任一信号肽的能力相关。 SecA 易位 ATP 酶或体外蛋白质易位，认为与 SecA64 的相互作用具有功能意义。因此，SecA64 提供了一个可溶的、明确的系统来研究 SecA 识别信号肽的模式以及信号肽释放的调节。
The selective recognition of pre-secretory proteins by SecA is essential to the process of protein export from Escherichia coli, yet very little is known about the requirements for recognition and the mode of binding of precursors to SecA, The major reason for this is the lack of a soluble system suitable for biophysical study of the SecA-precursor complex. Complicating the development of such a system is the likelihood that SecA interacts with the precursor in a high affinity, productive manner only when it is activated by binding to membrane and SecYEG. A critical aspect of the precursor/SecA interaction is that it is regulated by various SecA ligands (nucleotide, lipid, SecYEG) to facilitate the release of the precursor, most likely in a stepwise fashion, for translocation, Several recent reports show that functions of SecA can be studied using separated domains. Using this approach, we have isolated a proteolytically generated N-terminal fragment of SecA, which is stably folded, has high ATPase activity, and represents an activated version of SecA, We report here that this fragment, termed SecA64, binds signal peptides with significantly higher affinity than does SecA, Moreover, the ATPase activity of SecA64 is inhibited by signal peptides to an extent that correlates with the ability of these signal peptides to inhibit either SecA translocation ATPase or in vitro protein translocation, arguing that the interaction with SecA64 is functionally significant. Thus, SecA64 offers a soluble, well defined system to study the mode of recognition of signal peptides by SecA and the regulation of signal peptide release.