Crystal structures of an intrinsically active cholera toxin mutant yield insight into the toxin activation mechanism
Crystal structures of an intrinsically active cholera toxin mutant yield insight into the toxin activation mechanism
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DOI：
10.1021/bi0360152
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发表时间：
2004-04-06
期刊：
影响因子：
2.9
通讯作者：
Hol, WGJ
中科院分区：
文献类型：
--
作者：
O'Neal, CJ;Amaya, EI;Hol, WGJ
霍乱毒素（Cholera toxin，CT）是一种异源六聚体细菌蛋白毒素，属于A/B ADP核糖基化毒素大家族。这些毒素中的每一种都经历有限的蛋白水解和/或二硫键还原以形成酶活性毒性片段。切口和还原使得CT和来自大肠杆菌（LT）的密切相关的热不稳定肠毒素在溶液中不稳定，因此迄今为止阻止了对由毒素活化引起的构象变化的完整结构理解。我们提出了第一个结构一瞥的活性CT的结构，从三个晶体形式的一个单一的网站A-亚基CT的变体，Y30 S，它不需要激活的修改，充分的活动。我们还从两种晶体形式重新确定了野生型酶原CT的结构，这两种晶体形式都表现出（i）更好的几何形状和（ii）与先前确定的结构不同的A2“尾”构象[Zhang等人（1995）J. Mol. 251，563-573]。通过分析LT A-亚基R7 K变体的结构，在A-亚基环区域中观察到野生型CT和活性CTY 30 S之间的差异，所述A-亚基环区域先前与活化有关[货车den Akker et al.（1995）Biochemistry 34，10996-11004]。25-36活化环在CTY 30 S中是无序的，而47-56活性位点环在三种CTY 30 S结构中显示不同程度的有序，表明活化环中的无序使活性位点环比未活化的野生型CT中发现的更大程度的灵活性。在这六个新的观点的CT全毒素的基础上，我们提出了一个模型，野生型CT所经历的活化修饰是如何传递到活性位点的。
Cholera toxin (CT) is a heterohexameric bacterial protein toxin belonging to a larger family of A/B ADP-ribosylating toxins. Each of these toxins undergoes limited proteolysis and/or disulfide bond reduction to form the enzymatically active toxic fragment. Nicking and reduction render both CT and the closely related heat-labile enterotoxin from Escherichia coli (LT) unstable in solution, thus far preventing a full structural understanding of the conformational changes resulting from toxin activation. We present the first structural glimpse of an active CT in structures from three crystal forms of a single-site A-subunit CT variant, Y30S, which requires no activational modifications for full activity. We also redetermined the structure of the wild-type, proenzyme CT from two crystal forms, both of which exhibit (i) better geometry and (ii) a different A2 "tail" conformation than the previously determined structure [Zhang et al. (1995) J. Mol. Biol. 251, 563-573]. Differences between wild-type CT and active CTY30S are observed in A-subunit loop regions that had been previously implicated in activation by analysis of the structure of an LT A-subunit R7K variant [van den Akker et al. (1995) Biochemistry 34, 10996-11004]. The 25-36 activation loop is disordered in CTY30S, while the 47-56 active site loop displays varying degrees of order in the three CTY30S structures, suggesting that disorder in the activation loop predisposes the active site loop to a greater degree of flexibility than that found in unactivated wild-type CT. On the basis of these six new views of the CT holotoxin, we propose a model for how the activational modifications experienced by wild-type CT are communicated to the active site.