Core-Structural Evaluation of Protein-Protein Interfaces

本计划项目申请中计划的生物学实验将解决定义特定信号蛋白在细胞存活和迁移中的作用的重要问题。为了实现关键信号事件的分子基础的完整理解，几个关键的蛋白质-蛋白质相互作用的特点是通过一个综合的方法，涉及结晶学和核磁共振（NMR）光谱。该核心在该计划中的作用是识别功能复合物中伴侣分子的结合界面，并定义介导分子识别的接触表面的分子相互作用。通过直接观察与Rac的分子间接触，将阐明DOCK 180和GTdR Rac的结构，以了解DOCK 180的DHR-2结构域的鸟嘌呤核苷酸交换活性。假定的交换因子之间的相互作用 SHEP 1和对接蛋白p130 Cas将被定义，专注于分子功能，在细胞存活/迁移和Ras蛋白活性的调制的共同调节。还将阐明促凋亡复合物的结构特征，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NMR使用例如交叉饱和或SEA-TROSY实验进行分析。结构研究将验证研究者在各个组成部分中得出的假设。分子的细节将代表有价值的起点，设计特定的抑制剂和潜在的治疗，旨在破坏这些复合物在特定的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.