Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Ljubljana, Slovenia.
Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.
J Biol Chem. 2022 Oct;298(10):102455. doi: 10.1016/j.jbc.2022.102455. Epub 2022 Sep 3.
Pore-forming proteins perforate lipid membranes and consequently affect their integrity and cell fitness. Therefore, it is not surprising that many of these proteins from bacteria, fungi, or certain animals act as toxins. While pore-forming proteins have also been found in plants, there is little information about their molecular structure and mode of action. Bryoporin is a protein from the moss Physcomitrium patens, and its corresponding gene was found to be upregulated by various abiotic stresses, especially dehydration, as well as upon fungal infection. Based on the amino acid sequence, it was suggested that bryoporin was related to the actinoporin family of pore-forming proteins, originally discovered in sea anemones. Here, we provide the first detailed structural and functional analysis of this plant cytolysin. The crystal structure of monomeric bryoporin is highly similar to those of actinoporins. Our cryo-EM analysis of its pores showed an actinoporin-like octameric structure, thereby revealing a close kinship of proteins from evolutionarily distant organisms. This was further confirmed by our observation of bryoporin's preferential binding to and formation of pores in membranes containing animal sphingolipids, such as sphingomyelin and ceramide phosphoethanolamine; however, its binding affinity was weaker than that of actinoporin equinatoxin II. We determined bryoporin did not bind to major sphingolipids found in fungi or plants, and its membrane-binding and pore-forming activity was enhanced by various sterols. Our results suggest that bryoporin could represent a part of the moss defense arsenal, acting as a pore-forming toxin against membranes of potential animal pathogens, parasites, or predators.
孔形成蛋白在脂质膜上穿孔，从而影响其完整性和细胞适应性。因此，许多来自细菌、真菌或某些动物的这些蛋白作为毒素也就不足为奇了。虽然在植物中也发现了孔形成蛋白，但关于它们的分子结构和作用模式的信息却很少。Bryoporin 是来自藓类植物 Physcomitrium patens 的一种蛋白，其相应的基因被发现受到各种非生物胁迫的上调，特别是脱水以及真菌感染。基于氨基酸序列，它被认为与 actinoporin 家族的孔形成蛋白有关，该家族最初在海葵中发现。在这里，我们提供了对这种植物细胞溶素的首次详细结构和功能分析。单体 bryoporin 的晶体结构与 actinoporins 非常相似。我们对其孔的 cryo-EM 分析显示出 actinoporin 样的八聚体结构，从而揭示了进化上相距遥远的生物体中蛋白的密切亲缘关系。这进一步得到了我们的观察结果的证实，即 bryoporin 优先结合并在含有动物鞘脂的膜中形成孔，如鞘磷脂和神经酰胺磷酸乙醇胺；然而，它的结合亲和力比 actinoporin equinatoxin II 弱。我们确定 bryoporin 不与真菌或植物中发现的主要鞘脂结合，其膜结合和孔形成活性被各种甾醇增强。我们的结果表明， bryoporin 可能代表藓防御武器库的一部分，作为一种针对潜在动物病原体、寄生虫或捕食者的膜穿孔毒素。
