Order-disorder-order transitions mediate the activation of cholera toxin
Order-disorder-order transitions mediate the activation of cholera toxin
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DOI：
10.1016/j.jmb.2007.12.075
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发表时间：
2008-03-28
影响因子：
5.6
通讯作者：
Legge, Glen B.
中科院分区：
文献类型：
--
作者：
Ampapathi, Ravi S.;Creath, Andrea L.;Legge, Glen B.
霍乱毒素（CT）全毒素必须被激活以使宿主细胞中毒。该过程需要酶促CTA 1结构域从全毒素组分CTA 2和B5的细胞内解离，随后与宿主因子ADP核糖基化因子6（ARF 6）-GTP相互作用。我们报告的第一个基于NMR的解决方案的CT酶结构域（CTA 1）的结构数据。我们表明，这个自由的酶结构域部分展开的C-末端，并结合其蛋白质的合作伙伴在开始和结束这一激活过程。与无规卷曲化学位移（Delta Delta（coil））的偏差表明激活环中的螺旋形成，这对于打开毒素的活性位点至关重要，并且在其与人类蛋白质ARF 6结合之前发生。我们对游离的CTA 1和活性CTA 1：ARF 6-GTP与NAD（+）的复合物进行了NMR滴定，结果表明复合物的形成不会显著增强NAD（+）结合。通过使用4，4 '-双（1-苯胺基萘8-磺酸盐）荧光作为游离酶暴露的疏水特性的指示剂，进一步说明了CTA 1的部分解折叠，当与ARF 6-GTP结合时，游离酶的疏水特性显著降低。我们认为ARF 6变构的主要作用是诱导CTA 1的C-末端重折叠。因此，作为折叠的球状毒素复合物，CTA 1在胞质溶胶中逃脱内质网相关降解途径的伴侣蛋白和蛋白酶体组分，然后继续ADP核糖基化其靶G（s）α，触发与霍乱病理生理学相关的下游事件。(C)2007爱思唯尔有限公司保留所有权利。
Cholera toxin (CT) holotoxin must be activated to intoxicate host cells. This process requires the intracellular dissociation of the enzymatic CTA1 domain from the holotoxin components CTA2 and B5, followed by subsequent interaction with the host factor ADP ribosylation factor 6 (ARF6)-GTP. We report the first NMR-based solution structural data for the CT enzymatic domain (CTA1). We show that this free enzymatic domain partially unfolds at the C-terminus and binds its protein partners at both the beginning and the end of this activation process. Deviations from random coil chemical shifts (Delta delta(coil)) indicate helix formation in the activation loop, which is essential to open the toxin's active site and occurs prior to its association with human protein ARF6. We performed NMR titrations of both free CTA1 and an active CTA1:ARF6-GTP complex with NAD(+), which revealed that the formation of the complex does not significantly enhance NAD(+) binding. Partial unfolding of CTA1 is further illustrated by using 4,4'-bis(1-anilinonaphthalene 8-sulfonate) fluorescence as an indicator of the exposed hydrophobic character of the free enzyme, which is substantially reduced when bound to ARF6-GTP. We propose that the primary role of ARF6's allostery is to induce refolding of the C-terminus of CTA1. Thus, as a folded globular toxin complex, CTA1 escapes the chaperone and proteasomal components of the endoplasmic reticulum associated degradation pathway in the cytosol and then proceeds to ADP ribosylate its target G(s)alpha triggering the downstream events associated with the pathophysiology of cholera. (C) 2007 Elsevier Ltd. All rights reserved.