Pore formation by equinatoxin, a eukaryotic pore-forming toxin, requires a flexible N-terminal region and a stable β-sandwich
Pore formation by equinatoxin, a eukaryotic pore-forming toxin, requires a flexible N-terminal region and a stable β-sandwich
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DOI：
10.1074/jbc.m406193200
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发表时间：
2004-11-05
影响因子：
4.8
通讯作者：
Anderluh, G
中科院分区：
文献类型：
--
作者：
Kristan, K;Podlesek, Z;Anderluh, G
放线素是一种真核成孔蛋白，通过四种单体的自结合在天然和模型脂质膜上形成2纳米的孔。经历构象变化并构成跨膜孔一部分的区域目前正在被定义。最近的研究表明，equinatoxin的n端区域（残基10-28）参与了最终孔壁的构建。假设孔仅由多肽链形成，毒素的其他部分应该构成导电通道，在这里我们通过二硫扫描诱变来寻找这些区域。只有双半胱氨酸突变体的n端片段1-30与β -三明治连接，在二硫化物形成时溶血活性降低，这表明equinatoxin的其他部分，特别是β -三明治和重要的c端α -螺旋，在孔形成过程中没有发生大的构象重排。β -三明治稳定性的作用是通过隐藏的Trp突变使其部分疏水核心不稳定而独立评估的（117）。这些突变体的稳定性明显低于野生型，但表现出相似或略低的渗透活性。这些结果表明，灵活的n端区域和稳定的β -三明治是放线菌蛋白家族适当形成孔的先决条件。
Actinoporins are eukaryotic pore-forming proteins that create 2-nm pores in natural and model lipid membranes by the self-association of four monomers. The regions that undergo conformational change and form part of the transmembrane pore are currently being defined. It was shown recently that the N-terminal region (residues 10-28) of equinatoxin, an actinoporin from Actinia equina, participates in building of the final pore wall. Assuming that the pore is formed solely by a polypeptide chain, other parts of the toxin should constitute the conductive channel and here we searched for these regions by disulfide scanning mutagenesis. Only double cysteine mutants where the N-terminal segment 1-30 was attached to the beta-sandwich exhibited reduced hemolytic activity upon disulfide formation, showing that other parts of equinatoxin, particularly the beta-sandwich and importantly the C-terminal alpha-helix, do not undergo large conformational rearrangements during the pore formation. The role of the beta-sandwich stability was independently assessed via destabilization of a part of its hydrophobic core by mutations of the buried Trp(117). These mutants were considerably less stable than the wild-type but exhibited similar or slightly lower permeabilizing activity. Collectively these results show that a flexible N-terminal region and stable beta-sandwich are pre-requisite for proper pore formation by the actinoporin family.