Department of Chemistry, Tokyo Institute of Technology, 2-12-1 Meguro-ku, O-okayama, Tokyo 152-8551, Japan.
ACS Chem Biol. 2020 Jul 17;15(7):1808-1812. doi: 10.1021/acschembio.0c00403. Epub 2020 Jul 7.
Adenylation domains (A-domains) are responsible for selective incorporation of carboxylic acid substrates in the biosynthesis of various natural products. Each A-domain must recognize a cognate carrier protein (CP) for functional substrate transfer. The transient interactions between an A-domain and CP have been investigated by using acyl vinylsulfonamide adenosine inhibitors as probes to determine the structures of several A-domain-CP complexes. However, this strategy requires a specific vinylsulfonamide inhibitor that contains an acyl group corresponding to the substrate specificity of a target A-domain in every case. Here, we report an alternative strategy for structural characterization of A-domain-CP complexes. We used a bromoacetamide pantetheine cross-linking probe in combination with a Cys mutation to trap the standalone A-domain-CP complex involved in macrolactam polyketide biosynthesis through a covalent linkage, allowing the determination of the complex structure. This strategy facilitates the structural determination of A-domain-CP complexes.
腺苷酰化结构域（A 结构域）负责各种天然产物生物合成中羧酸底物的选择性掺入。每个 A 结构域都必须识别同源载体蛋白（CP）以进行功能性底物转移。通过使用酰基乙烯基砜酰胺腺苷抑制剂作为探针来研究 A 结构域和 CP 之间的瞬时相互作用，以确定几个 A 结构域-CP 复合物的结构。然而，该策略需要在每种情况下都含有与靶标 A 结构域的底物特异性相对应的酰基基团的特定乙烯基砜酰胺抑制剂。在这里，我们报告了一种用于 A 结构域-CP 复合物结构表征的替代策略。我们使用溴乙酰胺 pantetheine 交联探针与 Cys 突变相结合，通过共价键捕获涉及大环内酯聚酮生物合成的独立 A 结构域-CP 复合物，从而确定复合物的结构。该策略有助于 A 结构域-CP 复合物的结构测定。
