The proline-rich antimicrobial peptide (PrAMP) drosocin is produced by Drosophila species to combat bacterial infection. Unlike many PrAMPs, drosocin is O-glycosylated at threonine 11, a post-translation modification that enhances its antimicrobial activity. Here we demonstrate that the O-glycosylation not only influences cellular uptake of the peptide but also interacts with its intracellular target, the ribosome. Cryogenic electron microscopy structures of glycosylated drosocin on the ribosome at 2.0–2.8-Å resolution reveal that the peptide interferes with translation termination by binding within the polypeptide exit tunnel and trapping RF1 on the ribosome, reminiscent of that reported for the PrAMP apidaecin. The glycosylation of drosocin enables multiple interactions with U2609 of the 23S rRNA, leading to conformational changes that break the canonical base pair with A752. Collectively, our study reveals novel molecular insights into the interaction of O-glycosylated drosocin with the ribosome, which provide a structural basis for future development of this class of antimicrobials.

富含脯氨酸的抗菌肽 (PrAMP) 果蝇素由果蝇物种产生，用于对抗细菌感染。与许多 PrAMP 不同，卓索辛在苏氨酸 11 处进行 O-糖基化，这是一种增强其抗菌活性的翻译后修饰。在这里，我们证明 O-糖基化不仅影响肽的细胞摄取，而且还与其细胞内靶标核糖体相互作用。核糖体上糖基化卓索辛的低温电子显微镜结构以 2.0–2.8-Å 分辨率显示，该肽通过在多肽出口通道内结合并将 RF1 捕获在核糖体上来干扰翻译终止，这让人想起报道的 PrAMP apidaecin。果酱素的糖基化能够与 23S rRNA 的 U2609 发生多重相互作用，从而导致构象变化，破坏与 A752 的规范碱基对。总的来说，我们的研究揭示了 O-糖基化卓索辛与核糖体相互作用的新分子见解，为此类抗菌药物的未来开发提供了结构基础。