Data_Sheet_1_Dodecapeptide Cathelicidins of Cetartiodactyla: Structure, Mechanism of Antimicrobial Action, and Synergistic Interaction With Other Cathelicidins.docx

In this study, dodecapeptide cathelicidins were shown to be widespread antimicrobial peptides among the Cetruminantia clade. In particular, we investigated the dodecapeptide from the domestic goat Capra hircus, designated as ChDode and its unique ortholog from the sperm whale Physeter catodon (PcDode). ChDode contains two cysteine residues, while PcDode consists of two dodecapeptide building blocks and contains four cysteine residues. The recombinant analogs of the peptides were obtained by heterologous expression in Escherichia coli cells. The structures of the peptides were studied by circular dichroism (CD), FTIR, and NMR spectroscopy. It was demonstrated that PcDode adopts a β-hairpin structure in water and resembles β-hairpin antimicrobial peptides, while ChDode forms a β-structural antiparallel covalent dimer, stabilized by two intermonomer disulfide bonds. Both peptides reveal a significant right-handed twist about 200 degrees per 8 residues. In DPC micelles ChDode forms flat β-structural tetramers by antiparallel non-covalent association of the dimers. The tetramers incorporate into the micelles in transmembrane orientation. Incorporation into the micelles and dimerization significantly diminished the amplitude of backbone motions of ChDode at the picosecond-nanosecond timescale. When interacting with negatively charged membranes containing phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), the ChDode peptide adopted similar oligomeric structure and was capable to form ion-conducting pores without membrane lysis. Despite modest antibacterial activity of ChDode, a considerable synergistic effect of this peptide in combination with another goat cathelicidin – the α-helical peptide ChMAP-28 was observed. This effect is based on an increase in permeability of bacterial membranes. In turn, this mechanism can lead to an increase in the efficiency of the combined action of the synergistic pair ChMAP-28 with the Pro-rich peptide mini-ChBac7.5Nα targeting the bacterial ribosome.

本研究证实，十二肽类cathelicidin抗菌肽（cathelicidins）广泛分布于鲸反刍动物（Cetruminantia）演化支中。具体而言，本研究针对家山羊（Capra hircus）来源的十二肽（dodecapeptide）展开探究，将其命名为ChDode，同时也对来自抹香鲸（Physeter catodon）的独特直系同源物PcDode进行了分析。ChDode含有2个半胱氨酸残基，而PcDode则由2个十二肽结构单元构成，且含有4个半胱氨酸残基。本研究通过在大肠杆菌（Escherichia coli）细胞中进行异源表达，成功获得了两种肽的重组类似物。本研究通过圆二色谱（circular dichroism, CD）、傅里叶变换红外光谱（FTIR）与核磁共振（NMR）光谱学手段对两种肽的结构进行了解析。研究证实，PcDode在水溶液中可形成β发夹（β-hairpin）结构，与典型的β发夹类抗菌肽结构高度相似；而ChDode则形成由两条单体间二硫键稳定的β结构反平行共价二聚体。两种肽均展现出显著的右手扭曲特征，每8个残基的扭曲角度约为200度。在十二烷基磷酸胆碱（DPC）胶束体系中，ChDode通过二聚体的反平行非共价结合，形成扁平的β结构四聚体。该四聚体以跨膜取向嵌入胶束内部。嵌入胶束与二聚化过程，显著降低了ChDode在皮秒-纳秒（picosecond-nanosecond）时间尺度下的主链运动幅度。当与含有磷脂酰乙醇胺（phosphatidylethanolamine, PE）与磷脂酰甘油（phosphatidylglycerol, PG）的负电荷细菌膜相互作用时，ChDode可形成类似的寡聚体结构，并能够在不引发膜裂解的情况下构建离子传导孔道。尽管ChDode的抗菌活性较为温和，但本研究观察到，该肽与另一种山羊cathelicidin抗菌肽——α-螺旋（α-helical）肽ChMAP-28联合使用时，可产生显著的协同效应。该协同效应的核心机制为提升细菌细胞膜的通透性；而这一机制可进一步增强协同组合ChMAP-28与靶向细菌核糖体（ribosome）的富含脯氨酸（Pro-rich）肽mini-ChBac7.5Nα的联合作用效率。