Protein-Protein Interaction in Natural Product Biosynthesis

描述(申请人提供)：这个项目研究蛋白质-蛋白质相互作用在天然产物模块合成酶中的作用，这是一组三种类型的生物合成酶：聚酮合成酶(PKS)、非核糖体多肽合成酶(NRPS)和脂肪酸合成酶(FAS)。这些天然产品中，许多用作抗癌剂或抗生素，而另一些则是致病毒素。最近的证据表明，这些途径的载体蛋白结构域和催化结构域之间的蛋白质-蛋白质相互作用是正确催化和加工的关键。这些关键的相互作用本质上是暂时的，在很大程度上仍未确定其特征。我们已经开发了可以对载体蛋白结构域进行人工修饰的工具，在这里，我们利用这些工具通过底物模拟和共价交联抑制剂来延长这些蛋白质-蛋白质相互作用的寿命。这些工具将使我们能够直观地了解载体蛋白在催化前结合被束缚底物的分子细节，以及它们如何与同源催化结构域相互作用。通过这些研究，我们打算通过分析溶液相核磁共振谱来更全面地了解这些催化事件的动力学。修饰载体蛋白的核磁共振微扰比较和存在催化结构域的滴定实验将为我们提供一种能力 准确定位底物隔离和蛋白质-蛋白质相互作用所涉及的残基。我们将通过定点突变、氢/氚交换核磁共振和等温滴定量热法进一步验证我们的发现。对这些蛋白质-蛋白质相互作用的深入了解将影响药物发现的新兴领域。使用这些工具来指导新的相互作用将使新的药效团的组合生物合成成为可能，而通过取消病原体生物合成中的这些相互作用，可能会发现新的药物靶点。

DESCRIPTION (provided by applicant): This program investigates the role of protein-protein interactions in natural product modular synthases, a group of biosynthetic enzymes of three types: polyketide synthase (PKS), non-ribosomal peptide synthetase (NRPS), and fatty acid synthase (FAS). Many of these natural products serve as anti-cancer agents or antibiotics, while others are pathogenic toxins. Recent evidence indicates that protein-protein interactions between the carrier protein domain and catalytic domains of these pathways are key to proper catalysis and processivity. These critical interactions are transient in nature and remain largely uncharacterized. We have developed tools that allow synthetic modification of carrier protein domains, and here we leverage these tools to increase the lifetime of these protein-protein interactions using substrate mimics and covalent cross-linking inhibitors. These tools will allow us to visualize the molecular details by which carrier proteins bind tethered substrates prior to catalysis and how they interact with cognate catalytic domains. Through these studies, we intend to gain a more complete understanding of the dynamics of these catalytic events through analysis of solution phase NMR spectra. NMR perturbation comparisons of modified carrier proteins and titration experiments in the presence of catalytic domains will offer us an ability to accurately pinpoint the residues involved in substrate sequestration and protein-protein interaction. We will further validate our findings through site-directed mutagenesis, hydrogen/deuterium exchange NMR, and isothermal titration calorimetry. A deeper understanding of these protein-protein interactions will affect emerging fields in drug discovery. Using these tools to guide new interactions will enable combinatorial biosynthesis of new pharmacophores, while new drug targets may be discovered through abrogating these interactions in pathogen biosynthesis.