Heberlig Graham W, La Clair James J, Burkart Michael D
Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA, USA.
Nature. 2025 Feb;638(8049):261-269. doi: 10.1038/s41586-024-08306-y. Epub 2024 Dec 11.
Non-ribosomal peptide synthetases are assembly line biosynthetic pathways that are used to produce critical therapeutic drugs and are typically arranged as large multi-domain proteins called megasynthetases. They synthesize polypeptides using peptidyl carrier proteins that shuttle each amino acid through modular loading, modification and elongation steps, and remain challenging to structurally characterize, owing in part to the inherent dynamics of their multi-domain and multi-modular architectures. Here we have developed site-selective crosslinking probes to conformationally constrain and resolve the interactions between carrier proteins and their partner enzymatic domains. We apply tetrazine click chemistry to trap the condensation of two carrier protein substrates within the active site of the condensation domain that unites the first two modules of tyrocidine biosynthesis and report the high-resolution cryo-EM structure of this complex. Together with the X-ray crystal structure of the first carrier protein crosslinked to its epimerization domain, these structures highlight captured intermodular recognition events and define the processive movement of a carrier protein from one catalytic step to the next. Characterization of these structural relationships remains central to understanding the molecular details of these unique synthetases and critically informs future synthetic biology design of these pathways.
非核糖体肽合成酶是用于生产关键治疗药物的装配线生物合成途径，通常排列成称为巨型合成酶的大型多结构域蛋白质。它们使用肽基载体蛋白合成多肽，这些载体蛋白通过模块化加载、修饰和延伸步骤穿梭每个氨基酸，并且由于其多结构域和多模块结构的固有动态性，在结构表征方面仍然具有挑战性。在这里，我们开发了位点选择性交联探针，以构象约束和解析载体蛋白与其伙伴酶结构域之间的相互作用。我们应用四嗪点击化学来捕获两个载体蛋白底物在连接短杆菌酪肽生物合成前两个模块的缩合结构域活性位点内的缩合反应，并报告该复合物的高分辨率冷冻电镜结构。连同第一个载体蛋白与其差向异构化结构域交联的X射线晶体结构，这些结构突出了捕获的模块间识别事件，并定义了载体蛋白从一个催化步骤到下一个催化步骤的连续运动。这些结构关系的表征仍然是理解这些独特合成酶分子细节的核心，并为这些途径未来的合成生物学设计提供关键信息。
