Franzoi Marco, van Heuvel Yasemin, Thomann Susanne, Schürch Nadia, Kallio Pauli T, Venier Paola, Essig Andreas
Department of Biology, University of Padova , Via Ugo Bassi 58/B, Padova 35131, Italy.
Institute of Microbiology, Swiss Federal Institute of Technology, ETH Zurich , CH-8093 Zurich, Switzerland.
Biochemistry. 2017 Sep 19;56(37):4992-5001. doi: 10.1021/acs.biochem.7b00697. Epub 2017 Sep 6.
Defensins make up a class of cysteine-rich antimicrobial peptides, expressed by virtually all eukaryotes as part of their innate immune response. Because of their unique mode of action and rapid killing of pathogenic microbes, defensins are considered promising alternatives to clinically applied antibiotics. Copsin is a defensin-like peptide, previously identified in the mushroom Coprinopsis cinerea. It exerts its activity against a range of Gram-positive bacteria by binding to the peptidoglycan precursor lipid II and prevention of proper cell wall formation. In this study, we present a new workflow for the generation, production, and activity-driven selection of copsin derivatives, based on their expression in Pichia pastoris. One hundred fifty-two single-amino acid mutants and combinations thereof allowed the identification of k-copsin, a peptide variant exhibiting significantly enhanced activity against Bacillus subtilis and Staphylococcus aureus. Furthermore, we performed in silico characterizations of membrane interactions of copsin and k-copsin, in the presence and absence of lipid II. The molecular dynamics data highlighted a high variability in lipid II binding, with a preference for the MurNAc moiety with 47 and 35% of the total contacts for copsin and k-copsin, respectively. Mutated amino acids were located in loop regions of k-copsin and shown to be crucial in the perturbation of the bacterial membrane. These structural studies provide a better understanding of how defensins can be developed toward antibacterial therapies less prone to resistance issues.
防御素是一类富含半胱氨酸的抗菌肽，几乎所有真核生物都会将其作为固有免疫反应的一部分进行表达。由于其独特的作用方式以及对致病微生物的快速杀灭作用，防御素被认为是临床应用抗生素的有前景的替代物。科普辛是一种类防御素肽，先前在真菌灰盖鬼伞中被鉴定出来。它通过与肽聚糖前体脂 II 结合并阻止细胞壁的正常形成，对一系列革兰氏阳性菌发挥活性。在本研究中，我们基于科普辛在毕赤酵母中的表达，提出了一种用于科普辛衍生物的生成、生产及活性驱动筛选的新工作流程。152 个单氨基酸突变体及其组合使得 k - 科普辛得以鉴定，k - 科普辛是一种对枯草芽孢杆菌和金黄色葡萄球菌表现出显著增强活性的肽变体。此外，我们对存在和不存在脂 II 时科普辛和 k - 科普辛的膜相互作用进行了计算机模拟表征。分子动力学数据突出了脂 II 结合的高度变异性，科普辛和 k - 科普辛分别有 47%和 35%的总接触对 MurNAc 部分具有偏好性。突变的氨基酸位于 k - 科普辛的环区域，并且显示出对细菌膜的扰动至关重要。这些结构研究有助于更好地理解如何将防御素开发成不易产生耐药问题的抗菌疗法。
