Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, Brazil.
Department of Science and Environment, Roskilde University, Roskilde, Denmark.
PLoS One. 2018 Aug 30;13(8):e0202981. doi: 10.1371/journal.pone.0202981. eCollection 2018.
Actinoporins sticholysin I and sticholysin II (St I, St II) are proposed to lyse model and biomembranes via toroidal pore formation by their N-terminal domain. Based on the hypothesis that peptide fragments can reproduce the structure and function of this domain, the behavior of peptides containing St I residues 12-31 (StI12-31), St II residues 11-30 (StII11-30), and its TOAC-labeled analogue (N-TOAC-StII11-30) was examined. Molecular modeling showed a good match with experimental structures, indicating amphipathic α-helices in the same regions as in the toxins. CD spectra revealed that the peptides were essentially unstructured in aqueous solution, acquiring α-helical conformation upon interaction with micelles and large unilamellar vesicles (LUV) of variable lipid composition. Fluorescence quenching studies with NBD-containing lipids indicated that N-TOAC-StII11-30's nitroxide moiety is located in the membranes polar head group region. Pyrene-labeled phospholipid inter-leaflet redistribution suggested that the peptides form toroidal pores, according to the mechanism of action proposed for the toxins. Binding occurred only to negatively charged LUV, indicating the importance of electrostatic interactions; in contrast the peptides bound to both negatively charged and zwitterionic micelles, pointing to a lesser influence of these interactions. In addition, differences between bilayers and micelles in head group packing and in curvature led to differences in peptide-membrane interaction. We propose that the peptides topography in micelles resembles that of the toxins in the toroidal pore. The peptides mimicked the toxins permeabilizing activity, St II peptides being more effective than StI12-31. To our knowledge, this is the first demonstration that differences in the toxins N-terminal amphipathic α-helix play a role in the difference between St I and St II activities.
肌动蛋白孔形成毒素溶血素 I 和溶血素 II（St I、St II）被认为通过其 N 端结构域形成环形孔来裂解模型和生物膜。基于肽片段可以再现该结构域的结构和功能的假设，研究了含有 St I 残基 12-31（StI12-31）、St II 残基 11-30（StII11-30）及其 TOAC 标记类似物（N-TOAC-StII11-30）的肽的行为。分子建模显示与实验结构具有良好的匹配性，表明在同一区域具有两亲性 α-螺旋。CD 光谱表明，这些肽在水溶液中基本上没有结构，与胶束和具有不同脂质组成的大单室脂质体（LUV）相互作用时会获得 α-螺旋构象。用含有 NBD 的脂质进行荧光猝灭研究表明，N-TOAC-StII11-30 的硝氧自由基部分位于膜极性头部基团区域。芘标记的磷脂双层内叶重新分布表明，根据毒素的作用机制，肽形成环形孔。结合仅发生在带负电荷的 LUV 上，表明静电相互作用的重要性；相比之下，肽结合带负电荷和两性离子胶束，表明这些相互作用的影响较小。此外，双层和胶束在头部基团堆积和曲率方面的差异导致肽-膜相互作用的差异。我们提出，肽在胶束中的拓扑结构类似于毒素在环形孔中的拓扑结构。肽模拟了毒素的通透性活性，St II 肽比 StI12-31 更有效。据我们所知，这是首次证明毒素 N 端两亲性 α-螺旋的差异在 St I 和 St II 活性之间起作用。
