Ishikawa Fumihiro, Kasai Shota, Kakeya Hideaki, Tanabe Genzoh
Faculty of Pharmacy, Kindai University, 3-4-1 Kowakae, Higashi-Osaka, Osaka, 577-8502, Japan.
Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo, Kyoto, 606-8501, Japan.
Chembiochem. 2017 Nov 16;18(22):2199-2204. doi: 10.1002/cbic.201700361. Epub 2017 Oct 11.
Structural and activity studies have revealed the dynamic and transient actions of carrier protein (CP) activity in primary and secondary metabolic pathways. CP-mediated interactions play a central role in nonribosomal peptide biosynthesis, as they serve as covalent tethers for amino acid and aryl acid substrates and enable the growth of peptide intermediates. Strategies are therefore required to study protein-protein interactions efficiently. Herein, we describe activity-based probes used to demonstrate the protein-protein interactions between aryl CP (ArCP) and aryl acid adenylation (A) domains as well as the substrate specificities of the aryl acid A domains. If coupled with in-gel fluorescence imaging, this strategy allows visualization of the protein-protein interactions required to recognize and transfer the substrate to the partner ArCP. This technique has potential for the analysis of protein-protein interactions within these biosynthetic enzymes at the molecular level and for use in the combinatorial biosynthesis of new nonribosomal peptides.
结构和活性研究揭示了载体蛋白（CP）活性在初级和次级代谢途径中的动态和瞬时作用。CP介导的相互作用在非核糖体肽生物合成中起着核心作用，因为它们作为氨基酸和芳酸底物的共价连接物，并促进肽中间体的生长。因此，需要有效研究蛋白质-蛋白质相互作用的策略。在此，我们描述了基于活性的探针，用于证明芳基CP（ArCP）与芳酸腺苷化（A）结构域之间的蛋白质-蛋白质相互作用以及芳酸A结构域的底物特异性。如果与凝胶内荧光成像相结合，该策略可以可视化识别底物并将其转移至伴侣ArCP所需的蛋白质-蛋白质相互作用。该技术在分子水平上分析这些生物合成酶内的蛋白质-蛋白质相互作用以及用于新非核糖体肽的组合生物合成方面具有潜力。
