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.
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