Abstract:
Carrier protein-dependent biosynthesis provides a thiotemplated format for the production of natural products. Within these pathways, many reactions display exquisite substrate selectivity, a regulatory framework proposed to be controlled by protein-protein interactions (PPIs). In Escherichia coli, unsaturated fatty acids are generated within the de novo fatty acid synthase by a chain length-specific interaction between the acyl carrier protein AcpP and the isomerizing dehydratase FabA. To evaluate PPI-based control of reactivity, interactions of FabA with AcpP bearing multiple sequestered substrates were analyzed through NMR titration and guided high-resolution docking. Through a combination of quantitative binding constants, residue-specific perturbation analysis, and high-resolution docking, a model for substrate control via PPIs has been developed. The in silico results illuminate the mechanism of FabA substrate selectivity and provide a structural rationale with atomic detail. Helix III positioning in AcpP communicates sequestered chain length identity recognized by FabA, demonstrating a powerful strategy to regulate activity by allosteric control. These studies broadly illuminate carrier protein-dependent pathways and offer an important consideration for future inhibitor design and pathway engineering.
摘要:
载体蛋白依赖性生物合成为天然产物的生产提供了硫代模板形式。在这些途径中,许多反应显示出精致的底物选择性,提出由蛋白质-蛋白质相互作用(PPI)控制的调控框架。在大肠杆菌中,不饱和脂肪酸通过酰基载体蛋白AcpP和异构化脱水酶FabA之间的链长特异性相互作用在从头脂肪酸合酶中产生。为了评估基于PPI的反应性控制,通过NMR滴定和引导的高分辨率对接分析了FabA与带有多个隔离底物的AcpP的相互作用。通过定量结合常数的组合,特定于残基的扰动分析,和高分辨率对接,已经开发了通过PPI控制底物的模型。计算机模拟结果阐明了FabA底物选择性的机理,并提供了具有原子细节的结构原理。AcpP中的HelixIII定位传达了FabA识别的隔离链长身份,展示了通过变构控制来调节活性的强大策略。这些研究广泛阐明了载体蛋白依赖性途径,并为未来的抑制剂设计和途径工程提供了重要的考虑因素。