Core-Structural Evaluation of Protein-Protein Interfaces

本计划项目申请中计划的生物实验将解决定义特定信号蛋白在细胞生存和迁移中的作用的重要问题。为了完全了解关键信号事件的分子基础，将使用一种综合的方法来表征几种关键的蛋白质-蛋白质相互作用，该方法涉及结晶学和核磁共振(核磁共振)光谱。这个核心在该计划中的作用是识别功能复合体中伙伴分子上的结合界面，并确定在接触面上调节分子识别的分子相互作用。通过直接观察DOCK180与Rac的分子间相互作用，阐明DOCK180的结构和GTP酶Rac，以了解DOCK180的DHR-2结构域的鸟核苷酸交换活性。假定的交换因子之间的相互作用 SHEP1和对接蛋白p130Cas将被定义，重点是在细胞生存/迁移和调节RAS蛋白活性的共同调节中服务的分子特征。促凋亡复合体的结构特征也将被阐明，包括新的线粒体蛋白Bitl和转录调节因子AES。感兴趣的蛋白质结构域将被大规模生产并结晶。蛋白质-蛋白质界面将在溶液中通过核磁共振进行分析，例如使用交叉饱和或SEA-TROSY实验。结构研究将验证研究人员在各个组成部分中得出的假设。分子细节将为设计特定的抑制剂和潜在的治疗药物提供有价值的起点，旨在破坏这些复合体在特定信号事件中的生理作用。

The biological experiments planned in this Program Project application will address important questions defining the role of specific signaling proteins in cell survival and migration. To achieve a complete understanding of the molecular basis for critical signaling events, several key protein-protein interactions will be characterized by an integrated approach involving both crystallography and nuclear magnetic resonance (NMR) spectroscopy. The role of this Core in the Program is to identify binding interfaces on partner molecules in functional complexes and to define the molecular interactions at the contact surfaces that mediate molecular recognition. The structure of DOCK180 and the GTPase Rac will be elucidated to understand the guanine nucleotide exchange activity of the DHR-2 domain of DOCK180 by directly observing the intermolecular contacts with Rac. The interactions between the putative exchange factor SHEP1 and the docking protein p130Cas will be defined, focusing on molecular features that serve in co-regulation of cell survival/migration and modulation of Ras protein activity. The structural features of the pro-apoptotic complex including the novel mitochondrial protein Bitl and the transcriptional regulator AES will also be elucidated. The protein domains of interest will be produced on a large scale and crystallized. Protein-protein interfaces will be analyzed in solution by NMR using, for example, cross saturation or SEA-TROSY experiments. The structural studies will verify hypotheses derived by investigators in the individual Components. The molecular details will represent valuable starting points for the design of specific inhibitors and potential therapeutics directed to disrupt the physiological role of these complexes in specific signaling events.