Sites of interaction of a precursor polypeptide on the export chaperone SecB mapped by site-directed spin labeling
Sites of interaction of a precursor polypeptide on the export chaperone SecB mapped by site-directed spin labeling
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DOI：
10.1016/j.jmb.2006.07.021
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发表时间：
2006-10-13
影响因子：
5.6
通讯作者：
Randall, Linda L.
中科院分区：
文献类型：
--
作者：
Crane, Jennine M.;Suo, Yuying;Randall, Linda L.
通过一般分泌系统将蛋白质输出到大肠杆菌的周质中要求所输送的多肽缺乏稳定折叠的三级结构。前体多肽在折叠之前的捕获通过与伴侣SecB的混杂结合来实现。SecB通过其与SecA（膜易位子的外周组分）的特异性结合将其配体递送至输出位点。在易位子处，配体从SecB传递到SecA，随后通过SecYEG通道。我们以前使用的定点自旋标记和电子顺磁共振光谱建立SecB和SecA之间的对接模型。在这里，我们报告使用相同的策略来映射生理配体的途径，前体半乳糖结合蛋白的未折叠形式，SecB。我们的一组SecB变体，每个变体包含一个单一的半胱氨酸，这是在以前的研究中使用的，已经扩大到48个残基，覆盖49%的SecB表面。参与接触的SecB上的残基被鉴定为在加入未折叠的多肽配体后显示出与氮氧自由基的受限运动一致的谱线形状变化的那些残基。我们的结论是，绑定前体接触的小分子伴侣的表面的大部分。SecB上与配体相互作用的网站进行了比较与先前确定的网站，与SecA相互作用的配体和转移的模型进行了讨论。(c)2006爱思唯尔有限公司保留所有权利。
Export of protein into the periplasm of Escherichia coli via the general secretory system requires that the transported polypeptides be devoid of stably folded tertiary structure. Capture of the precursor polypeptides before they fold is achieved by the promiscuous binding to the chaperone SecB. SecB delivers its ligand to export sites through its specific binding to SecA, a peripheral component of the membrane translocon. At the translocon the ligand is passed from SecB to SecA and subsequently through the SecYEG channel. We have previously used site-directed spin labeling and electron paramagnetic resonance spectroscopy to establish a docking model between SecB and SecA. Here we report use of the same strategy to map the pathway of a physiologic ligand, the unfolded form of precursor galactose-binding protein, on SecB. Our set of SecB variants each containing a single cysteine, which was used in the previous study, has been expanded to 48 residues, which cover 49% of the surface of SecB. The residues on SecB involved in contacts were identified as those that, upon addition of the unfolded polypeptide ligand, showed changes in spectral line shape consistent with restricted motion of the nitroxide. We conclude that the bound precursor makes contact with a large portion of the surface of the small chaperone. The sites on SecB that interact with the ligand are compared with the previously identified sites that interact with SecA and a model for transfer of the ligand is discussed. (c) 2006 Elsevier Ltd. All rights reserved.