Structural Insights into the Mode of Action of the Peptide Antibiotic Copsin

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（Defensins）是一类富含半胱氨酸的抗菌肽，几乎所有真核生物均会表达此类肽类，作为其先天免疫应答的组成部分。由于其独特的作用模式与快速杀灭病原微生物的能力，防御素被视为临床应用抗生素的极具潜力的替代方案。Copsin是一种类防御素肽，此前在灰盖鬼伞（Coprinopsis cinerea）中被发现。它可通过结合肽聚糖前体脂质II（lipid II）并阻碍正常细胞壁合成，对多种革兰氏阳性菌发挥抗菌活性。本研究基于毕赤酵母（Pichia pastoris）表达体系，开发了一套全新的工作流程，用于Copsin衍生物的构建、制备与活性筛选。通过152个单氨基酸突变体及其组合突变体的筛选，本研究获得了k-copsin这一肽变体，其对枯草芽孢杆菌（Bacillus subtilis）与金黄色葡萄球菌（Staphylococcus aureus）的抗菌活性显著提升。此外，本研究在有无脂质II的条件下，对Copsin与k-copsin的膜相互作用开展了计算机模拟表征。分子动力学数据显示，二者在脂质II结合模式上存在较高变异性，Copsin与k-copsin分别有47%与35%的总接触位点偏好结合MurNAc基团。突变氨基酸位点位于k-copsin的环区，且被证实对破坏细菌细胞膜具有关键作用。上述结构研究有助于更深入地理解防御素如何被开发为更不易产生耐药性的抗菌治疗手段。