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

项目概要/摘要 生物体的物理相互作用组，是由所有可能发生的物理相互作用形成的网络 在其所有大分子之间的生理相关动态范围内，包括蛋白质-蛋白质、DNA- 蛋白质，以及RNA-蛋白质相互作用，是让我们从全局角度理解细胞的关键层。 在这项资助申请中，我们将重点关注全球相互作用组网络的一个特定方面： 执行所有主要分子反应所需的分子机器或复合物。虽然蛋白质复合物 负责大多数生物过程，组织的全球模型和完整的 对于包括人类在内的大多数物种来说，蛋白质复合物或“复合体”的集合仍然非常不完整。我们 建议精确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。我们将系统地将所有开放的阅读框架碎片化 对应于编码酵母复合体的~300个复合体的所有~ 1，200个基因，并测试直接 使用四种互补蛋白质-蛋白质 相互作用（PPI）测定，一种基于Gal 4蛋白在酵母中的重构（Gal 4-Y2 H），另一种基于Gal 4蛋白在酵母中的重构（Gal 4-Y2 H）。 三个是基于在三种不同的表达系统中重建一种叫做NanoLuc的荧光素酶蛋白。 然后，我们将描述所识别的结构域-结构域相互作用的序列要求，首先- 将使用界面预测建模来生成复合物的生成模型。的长期愿景 这个建议是在每个复合物中产生二进制蛋白质相互作用信息，最终， 对于建立酵母复合体的完整结构非常有价值。这反过来又会使 结合蛋白质复合物在时间和空间上的动态方面， 基因型-表型关系模型。

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.