The equinatoxin N-terminus is transferred across planar lipid membranes and helps to stabilize the transmembrane pore
The equinatoxin N-terminus is transferred across planar lipid membranes and helps to stabilize the transmembrane pore
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DOI：
10.1111/j.1742-4658.2006.05608.x
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发表时间：
2007-01-01
期刊：
影响因子：
5.4
通讯作者：
Anderluh, Gregor
中科院分区：
文献类型：
--
作者：
Kristan, Katarina;Viero, Gabriella;Anderluh, Gregor
Equinatoxin II是一种从海葵Actinia equina中分离出来的溶细胞蛋白。它是actinoporins的一员，actinoporins是真核细胞成孔毒素家族，具有独特的成孔机制。马毒素II是一种20 kDa的无半胱氨酸蛋白，具有鞘磷脂依赖性活性。最近的研究表明，分子的N-末端区域在孔形成期间需要构象柔性。了解最终孔中的N-末端位置及其在膜插入和孔稳定性中的作用对于定义孔形成的精确分子机制是至关重要的。用平面脂质膜研究了孔的形成及其电生理特性。我们表明，氨基酸的位置1和3的马眼毒素II暴露于孔腔。此外，巯基试剂和一个六组氨酸标签连接到N-末端显示，毒素的N-末端延伸通过孔的其他（反）侧的膜和孔内的带负电荷的残基是至关重要的定义通道的电生理特性。最后，我们检测到一个新的，不太稳定的，具有较低的电导状态，通过使用删除突变体，其中前五个N-末端氨基酸被删除。我们认为前五个氨基酸有助于将两亲性螺旋锚在膜的反式侧，从而稳定最终的跨膜孔。
Equinatoxin II is a cytolytic protein isolated from the sea anemone Actinia equina. It is a member of the actinoporins, a family of eukaryotic pore-forming toxins with a unique mechanism of pore formation. Equinatoxin II is a 20 kDa cysteineless protein, with sphingomyelin-dependent activity. Recent studies showed that the N-terminal region of the molecule requires conformational flexibility during pore formation. An understanding of the N-terminal position in the final pore and its role in membrane insertion and pore stability is essential to define the precise molecular mechanism of pore formation. The formation of pores and their electrophysiologic characteristics were studied with planar lipid membranes. We show that amino acids at positions 1 and 3 of equinatoxin II are exposed to the lumen of the pore. Moreover, sulfhydryl reagents and a hexa-histidine tag attached to the N-terminus revealed that the N-terminus of the toxin extends through the pore to the other (trans) side of the membrane and that negatively charged residues inside the pore are crucial to define the electrophysiologic characteristics of the channel. Finally, we detected a new, less stable, state with a lower conductance by using a deletion mutant in which the first five N-terminal amino acids were removed. We propose that the first five amino acids help to anchor the amphipathic helix on the trans side of the membrane and consequently stabilize the final transmembrane pore.