Structural Characterization of Complex of Adenylation Domain and Carrier Protein by Using Pantetheine Cross-Linking Probe.,ACS Chemical Biology

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Structural Characterization of Complex of Adenylation Domain and Carrier Protein by Using Pantetheine Cross-Linking Probe.
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2020-07-01 , DOI: 10.1021/acschembio.0c00403
Akimasa Miyanaga ₁ , Shohei Kurihara ₁ , Taichi Chisuga ₁ , Fumitaka Kudo ₁ , Tadashi Eguchi ₁

Affiliation

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.

中文翻译：

番红碱交联探针对腺苷酸化域和载体蛋白复合物的结构表征。

腺苷酸化域（A域）负责在各种天然产物的生物合成中选择性掺入羧酸底物。每个A结构域必须识别功能载体底物转移的同源载体蛋白（CP）。通过使用酰基乙烯基磺酰胺腺苷抑制剂作为探针来确定几种A结构域-CP复合物的结构，已经研究了A结构域与CP之间的瞬时相互作用。然而，该策略需要特定的乙烯基磺酰胺抑制剂，其在每种情况下均包含对应于靶标A结构域的底物特异性的酰基。在这里，我们报告了A域CP复合物结构表征的替代策略。我们将溴乙酰胺泛肽交联探针与Cys突变结合使用，以通过共价键捕获参与大内酰胺聚酮化合物生物合成的独立A结构域-CP复合物，从而确定了复合物的结构。这种策略促进了结构域A域CP复合物的确定。

更新日期：2020-07-17

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