Kristan Katarina, Viero Gabriella, Macek Peter, Dalla Serra Mauro, Anderluh Gregor
Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.
FEBS J. 2007 Jan;274(2):539-50. doi: 10.1111/j.1742-4658.2006.05608.x.
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.
海葵毒素II是一种从海葵黄海葵中分离出的细胞溶解蛋白。它是放线孔蛋白家族的成员，该家族是一类真核生物成孔毒素，具有独特的成孔机制。海葵毒素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，该分子的N端区域在成孔过程中需要构象灵活性。了解最终孔中N端的位置及其在膜插入和孔稳定性中的作用对于确定精确的成孔分子机制至关重要。利用平面脂质膜研究了孔的形成及其电生理特性。我们发现海葵毒素II第1和第3位的氨基酸暴露于孔腔。此外，巯基试剂和连接到N端的六组氨酸标签表明，毒素的N端穿过孔延伸到膜的另一侧（反侧），并且孔内带负电荷的残基对于确定通道的电生理特性至关重要。最后，我们通过使用缺失前五个N端氨基酸的缺失突变体检测到一种新的、不太稳定的、电导率较低的状态。我们提出，前五个氨基酸有助于将两亲性螺旋锚定在膜的反侧，从而稳定最终的跨膜孔。
