Protein-Protein Interaction in Natural Product Biosynthesis

 描述（由申请人提供）：该计划研究蛋白质-蛋白质相互作用在天然产物模块化脱氢酶中的作用，模块化脱氢酶是一组三种类型的生物合成酶：聚酮合酶（PKS），非核糖体肽合成酶（NRPS）和脂肪酸合酶（FAS）。这些天然产物中的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伴侣蛋白之间的蛋白质-蛋白质相互作用是适当的催化和持续合成能力的关键。这些关键的相互作用本质上是短暂的，并且在很大程度上仍然没有特征。我们已经开发了允许载体蛋白结构域的合成修饰的工具，在这里，我们利用这些工具，使用底物模拟物和共价交联抑制剂来增加这些蛋白质-蛋白质相互作用的寿命。这些工具将 使我们能够可视化的分子细节载体蛋白结合拴系底物之前，催化和它们如何相互作用的同源催化合作伙伴。通过这些研究，我们打算获得一个更完整的了解这些催化事件的分子相互作用。我们将开发新的交联探针来捕获载体蛋白及其催化伙伴进入它们的结合状态。通过溶液相NMR光谱和X射线晶体学分析的后续结构研究将用于可视化交联物质。此外，在伴侣蛋白存在下的NMR滴定实验将进一步使我们能够准确地查明参与底物螯合和蛋白质-蛋白质相互作用的残基。我们将通过定点诱变、交联研究和其他生物物理方法进一步验证我们的发现。最后，我们将使用分子动力学计算沿着与结构生物学的数据，通过改变FAS和NRPS途径之间的蛋白质-蛋白质相互作用来设计一种新的代谢途径。对这些蛋白质-蛋白质相互作用以及如何控制它们的更深入理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药效团的组合生物合成成为可能，而新的药物靶点可以通过废除病原体生物合成中的这些相互作用来发现。

 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 partner proteins 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 partners. Through these studies, we intend to gain a more complete understanding of the molecular interactions of these catalytic events. We will develop new crosslinking probes to trap carrier proteins and their catalytic partners into their bound state. Subsequent structural studies through analysis of solution phase NMR spectra and X-ray crystallography will be used to visualize the crosslinked species. In addition, NMR titration experiments in the presence of partner proteins will further allow us to accurately pinpoint the residues involved in substrate sequestration and protein-protein interaction. We will further validate our findings through site-directed mutagenesis, crosslinking studies, and other biophysical methods. Finally, we will use molecular dynamics computation along with the data from structural biology to design a novel metabolic pathway through modification of protein-protein interactions between a FAS and NRPS pathway. A deeper understanding of these protein-protein interactions and how to control them 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.