Probing Transcriptional Activation at the Molecular Level

描述（由申请人提供）：转录激活因子、共激活因子及其复合物的构象动力学支持受调控的转录。单个构象之间的低能量屏障意味着每个参与者可以使用相同的氨基酸组来识别各种结合伴侣，每个复合物呈现不同的构象。假设是每个构象通过与其他结合伴侣的变构通信与独特的功能结果相关。然而，该模型从未经过测试，主要是由于缺乏将体外观察与细胞功能联系起来的强大工具。我们的目标是为此目的开发和实施化学遗传工具。专注于两个功能上重要的辅激活基序，KIX（神经性疼痛，神经退行性疾病）和AcID（癌症，病毒感染），我们将开发共价化学辅分子伴侣，稳定特定构象的辅激活单独和复杂的同源配体。这些辅助分子伴侣将用于严格表征不同构象和组装状态下辅助激活剂的结构（X射线晶体学、核磁共振光谱学）和动力学（瞬时动力学、平衡结合、计算分析）。补充在体外数据将与化学共分子伴侣在细胞中的实验，从而确定相互作用和构象，调节转录输出。通过这些研究，将确定这些疾病相关转录复合物的小分子靶向的变构结合位点。

DESCRIPTION (provided by applicant): The conformational dynamics of transcriptional activators, coactivators and their complexes underpin regulated transcription. The low energy barriers between individual conformations mean that each participant can use the same group of amino acids to recognize a variety of binding partners, with each complex assuming a distinct conformation. The assumption is that each of the conformations correlates with a unique functional outcome via allosteric communication with other binding partners. However, this model has never been tested, primarily due to a lack of robust tools to connect in vitro observations with function in cells. Our goal is to develop and implement chemical genetic tools for this purpose. Focusing on two functionally important coactivator motifs, KIX (neuropathic pain, neurodegenerative disorders) and AcID (cancer, viral infection), we will develop covalent chemical co-chaperones that stabilize particular conformations of the coactivators alone and in complex with cognate ligands. These co-chaperones will be used to rigorously characterize the structure (X-ray crystallography, NMR spectroscopy) and dynamics (transient kinetics, equilibrium binding, computational analysis) of the coactivators in distinct conformational and assembly states. Complementing the in vitro data will be experiments with chemical co-chaperones in cells, thus identifying both the interactions and the conformations that regulate transcriptional output. Through these studies, allosteric binding sites for small molecule targeting of these disease-relevant transcriptional complexes will be identified.