Protein-Protein Interactions in Natural Product Biosynthesis

7.项目总结。 这个项目研究了蛋白质之间的相互作用在载体蛋白质依赖的生物合成中的作用， 包括脂肪酸合成酶、聚酮合成酶和非核糖体肽合成酶途径。这个 与这些途径相关的天然产物可用作治疗药物，包括抗生素、抗真菌药物和 抗癌剂。指导这些途径的生物合成以生产未尝试过的 生物活性化合物对于新的和改进的治疗方法至关重要。在前几年，我们展示了 载体蛋白和伴侣蛋白结构域之间的蛋白质相互作用如何指导反应性和 引导过程性。此外，我们开发了一套化学生物学工具来稳定和审问这些 通过创新的交联剂开发和结构生物学实现原子分辨率的相互作用。我们现在 建议用新的笼式交联剂来扩展这些工具，这些交联剂旨在捕获瞬时伴侣蛋白质。 有了这个全面的交联剂文库，我们将评估载体蛋白与合作伙伴的相互作用 从脂肪酸和聚酮中提取的蛋白质，包括酮基还原酶、烯基还原酶和硫代酯酶 聚酮合成酶的合成酶、酮合成酶/链长因子和酰基转移酶，以及 非核糖体多肽合成酶的缩合结构域、卤代酶和氧化物酶。一旦被抓获， 这些交联物将通过溶液相核磁共振、动力学和热力学分析、X-射线分析进行研究。 结晶学和分子动力学模拟，以更全面地阐明机制和特异性 由载体蛋白-底物/中间体/ProDU相互作用产生。最后，我们将使用在此中收集的数据 计划作为一个信息平台，使合成生物生产新的天然产物杂交成为可能。

7. Project Summary. This program investigates the role of protein'protein interactions in carrier protein-dependent biosynthesis, including fatty acid synthase, polyketide synthase, and non-ribosomal peptide synthetase pathways. The natural products associated with these pathways serve as therapeutics including antibiotics, antifungals, and anticancer agents. The ability to direct the biosynthesis of these pathways for the production of untried bioactive compounds is of critical importance for new and improved therapies. In prior years, we demonstrated how protein'protein interactions between carrier proteins and partner protein domains direct reactivity and guide processivity. Moreover, we developed a suite of chemical biology tools to stabilize and interrogate these interactions at atomic resolution through innovative crosslinker development and structural biology. We now propose to expand these tools with new, caged crosslinkers designed to capture transient partner proteins. With this comprehensive library of crosslinkers, we will evaluate carrier protein interactions with partner proteins that include ketoreductases, enoyl reductases, and thioesterases from fatty acid and polyketide synthases, ketosynthase/chain-length factors and acyltransferases from polyketide synthases, and condensation domains, halogenases, and oxidases from non-ribosomal peptide synthetases. Once captured, these crosslinked species will be studied by solution-phase NMR, kinetic and thermodynamic assays, X-ray crystallography, and molecular dynamics simulations to more fully elucidate mechanism and specificity conferred by carrier protein-substrate/intermediate/produ interactions. Finally, we will use data collected in this program as an informatic platform to enable synthetic biological production of new natural product hybrids.