Many peptide hormones adopt long α-helical structures upon interacting with their cognate receptors but often exhibit flexible conformations when unbound. Strategies that can stabilize long α-helices without disrupting their binding to receptors are still lacking, which hinders progress in their biological applications and drug development. Here, we present an approach that combines rational design with library screening to create and identify a unique disulfide-directed multicyclic peptide (DDMP) scaffold, which could effectively stabilize N-terminally extendable α-helices while displaying exceptional efficiency in disulfide pairing and oxidative folding. This DDMP scaffold was then utilized for stabilizing the α-helical structure of glucagon-like peptide-1 (GLP-1), resulting in a potent GLP-1 receptor (GLP-1R) agonist with a significantly improved α-helicity and proteolytic stability. By incorporating external α-helices into the DDMP scaffold, we can effectively preserve the native N-terminal α-helical structures while allowing for extensive evolution of the C-terminal disulfide-rich domain for enhancing target binding, as demonstrated by the generation of the DDMP-stabilized GLP-1 (g1:Ox). The cryo-electron microscopy structure of the g1:Ox–GLP-1R in complex with heterotrimeric G_s reveals the molecular basis for the potent binding between g1:Ox and GLP-1R. Specifically, the DDMP moiety establishes additional interactions with the extracellular domain of GLP-1R, which are absent in the case of GLP-1. Thus, this work offers a novel and effective approach for engineering therapeutic peptides and other peptide α-helices, ensuring that both the N- and C-terminal regions remain essential for target recognition and activation.

中文翻译：

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具有 N 末端可延伸 α-螺旋的二硫键定向多环肽，用于识别和激活 G 蛋白偶联受体


许多肽激素在与其同源受体相互作用时采用长α螺旋结构，但在未结合时通常表现出灵活的构象。仍然缺乏能够在不破坏长α-螺旋与受体结合的情况下稳定其策略，这阻碍了其生物学应用和药物开发的进展。在这里，我们提出了一种将理性设计与文库筛选相结合的方法，以创建和鉴定一种独特的二硫键定向多环肽 （DDMP） 支架，它可以有效地稳定 N 末端可延伸的 α-螺旋，同时在二硫键配对和氧化折叠方面表现出卓越的效率。然后，该 DDMP 支架用于稳定胰高血糖素样肽-1 （GLP-1） 的α螺旋结构，从而产生有效的 GLP-1 受体 （GLP-1R） 激动剂，具有显着改善的 α-螺旋性和蛋白水解稳定性。通过将外部 α 螺旋掺入 DDMP 支架中，我们可以有效地保留天然 N 端α螺旋结构，同时允许富含 C 端二硫键的结构域的广泛进化以增强靶标结合，如 DDMP 稳定的 GLP-1 （g1：Ox） 的产生所证明的那样。g1：Ox-GLP-1R 与异源三聚体 G_s 复合物的冷冻电子显微镜结构揭示了 g1：Ox 和 GLP-1R 之间有效结合的分子基础。具体来说，DDMP 部分与 GLP-1R 的胞外结构域建立了额外的相互作用，而这些相互作用在 GLP-1 的情况下是不存在的。因此，这项工作为工程治疗性肽和其他肽 α 螺旋提供了一种新颖有效的方法，确保 N 端和 C 端区域对于靶标识别和激活仍然是必不可少的。