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 megasynthetases¹. They synthesize polypeptides using peptidyl carrier proteins that shuttle each amino acid through modular loading, modification and elongation² steps, and remain challenging to structurally characterize, owing in part to the inherent dynamics of their multi-domain and multi-modular architectures³. Here we have developed site-selective crosslinking probes to conformationally constrain and resolve the interactions between carrier proteins and their partner enzymatic domains^4,5. 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流水线生物合成途径，通常排列为称为巨合成酶¹ 的大型多结构域蛋白。他们使用肽基载体蛋白合成多肽，这些蛋白通过模块化加载、修饰和延伸² 个步骤穿梭每个氨基酸，并且在结构表征方面仍然具有挑战性，部分原因是其多结构域和多模块结构的固有动力学³。在这里，我们开发了位点选择联探针，以构象限制和解析载体蛋白与其伙伴酶结构域之间的相互作用^4,5。我们应用四嗪点击化学来捕获两个载体蛋白底物在结合酪氨酸生物合成前两个模块的缩合结构域的活性位点内的缩合，并报告了该复合物的高分辨率冷冻电镜结构。这些结构与第一个交联到其差向异构结构域的载体蛋白的 X 射线晶体结构一起，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过程运动。这些结构关系的表征仍然是理解这些独特合成酶的分子细节的核心，并为这些途径的未来合成生物学设计提供关键信息。