Interface-resolution domain-domain interactome map of the yeast complexome

项目摘要/摘要 有机体的物理互动体，它是由所有可能发生的物理相互作用形成的网络 在所有大分子之间的生理相关的动态范围内，包括蛋白质-蛋白质，DNA- 蛋白质，以及RNA-蛋白质相互作用，是让我们从全球角度理解细胞的关键一层。 在这项赠款申请中，我们将重点关注全球互动组网络的一个特定方面：所有 执行所有主要分子反应所需的分子机器或复合体。而蛋白质复合体 负责大多数生物过程、全球模型的组织和架构的完成 对于包括人类在内的大多数物种来说，蛋白质复合体或“复合体”仍然是极不完整的。我们 建议精确地映射酵母复合体亚基之间的直接结构域相互作用 分辨率接近接口级分辨率。我们将系统地碎片化所有开放阅读框架 与编码酵母复合体~300复合体的全部~1,200个基因相对应，并检测直接 利用四种互补蛋白-蛋白质在相应编码域之间的物理相互作用 相互作用(PPI)分析，一种基于Gal4蛋白在酵母中的重组(Gal4-Y2H)，另一种基于Gal4-Y2H 三是基于一种名为NanoLuc的荧光素酶蛋白在三种不同的表达系统中的重组。 然后，我们将表征已识别的域-域相互作用的序列要求，并首先- 将使用界面预测建模来生成络合物的生成模型。的长期愿景 这项提议是在每个复合体内产生二元蛋白质相互作用信息，最终将是 对建立酵母复合体的完整结构非常有价值。这反过来又将允许 融合蛋白质复合体在时间和空间上的动态特征并产生改进的预测 基因型-表型关系的模型。

Project Summary/Abstract The physical interactome of an organism, which is the network formed by all physical interactions that can occur in a physiologically relevant dynamic range between all its macromolecules, including protein-protein, DNA- protein, and RNA-protein interactions, is a critical layer to allow us to understand cells from a global point-view. In this grant application, we will focus on a particular aspect of global interactome networks: the formation of all molecular machines or complexes needed to execute all major molecular reactions. While protein complexes are responsible for most biological processes, global models of the organization and architecture of complete sets of protein complexes, or “complexomes”, are still vastly incomplete for most species, including human. We propose to precisely map direct domain-domain interactions between subunits of the yeast complexome with a resolution approaching interface-level resolution. We will systematically fragment all open reading frames corresponding to all ~1,200 genes encoding the ~300 complexes of the yeast complexome and test for direct physical interactions between the corresponding encoded domains using four complementary protein-protein interaction (PPI) assays, one based on the reconstitution of the Gal4 protein in yeast (Gal4-Y2H) and the other three based on the reconstitution of a luciferase protein called NanoLuc in three different expression systems. We will then characterize the sequence requirements of the identified domain-domain interactions and first- generation models of complexes will be generated using interface prediction modeling. The long-term vision of this proposal is to generate binary protein interaction information inside each complex, which, eventually, will be extremely valuable to establish the complete architecture of the yeast complexome. This in turn will allow incorporating dynamic aspects of protein complexes in time and space and generating improving predictive models of genotype-phenotype relationships.