Structural Basis of Clostridium perfringens Enterotoxin Activation and Oligomerization by Trypsin.
Structural Basis of Clostridium perfringens Enterotoxin Activation and Oligomerization by Trypsin.
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DOI：
10.3390/toxins15110637
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发表时间：
2023-10-31
期刊：
影响因子：
4.2
通讯作者：
Vecchio AJ
中科院分区：
文献类型：
--
作者：
Ogbu CP;Kapoor S;Vecchio AJ
产气荚膜梭菌肠毒素（CpE）是一种β-孔形成毒素，通过结合称为cladin的膜蛋白受体破坏哺乳动物胃肠道稳态。虽然已经确定了结合在cladin上的CpE片段的结构，但触发CpE活化和寡聚化导致细胞毒性β孔形成的机制仍未确定。胰蛋白酶对肠道内CpE的蛋白水解已被证明在这一过程和随后的细胞毒性过程中发挥作用。在这里，我们用小角x射线散射（SAXS）报道了全长和胰蛋白酶化CpE的溶液结构，用x射线晶体学报道了胰蛋白酶化CpE及其c端cludin结合域（cCpE）的晶体结构。质谱分析和SAXS发现胰蛋白酶去除CpE n端会改变CpE结构，从而暴露出通常未暴露的区域。胰蛋白酶化CpE和cCpE的晶体结构显示出独特的二聚体界面，可以作为寡聚化位点。此外，将这些结构与现有结构进行比较，可以预测在细胞受体结合和β孔形成的背景下寡聚化的功能含义。这项研究揭示了胰蛋白酶在改变CpE结构中所起的作用，通过诱导其在细胞毒性β孔形成的途径上的寡聚化来激活其功能。它的发现可以激发新的方法来抑制基于cpe的低聚物破坏疗法的细胞毒性。
Clostridium perfringens enterotoxin (CpE) is a β-pore forming toxin that disrupts gastrointestinal homeostasis in mammals by binding membrane protein receptors called claudins. Although structures of CpE fragments bound to claudins have been determined, the mechanisms that trigger CpE activation and oligomerization that lead to the formation of cytotoxic β-pores remain undetermined. Proteolysis of CpE in the gut by trypsin has been shown to play a role in this and subsequent cytotoxicity processes. Here, we report solution structures of full-length and trypsinized CpE using small-angle X-ray scattering (SAXS) and crystal structures of trypsinized CpE and its C-terminal claudin-binding domain (cCpE) using X-ray crystallography. Mass spectrometry and SAXS uncover that removal of the CpE N-terminus by trypsin alters the CpE structure to expose areas that are normally unexposed. Crystal structures of trypsinized CpE and cCpE reveal unique dimer interfaces that could serve as oligomerization sites. Moreover, comparisons of these structures to existing ones predict the functional implications of oligomerization in the contexts of cell receptor binding and β-pore formation. This study sheds light on trypsin’s role in altering CpE structure to activate its function via inducing oligomerization on its path toward cytotoxic β-pore formation. Its findings can incite new approaches to inhibit CpE-based cytotoxicity with oligomer-disrupting therapeutics.