Probing Transcriptional Activation at the Molecular Level - Diversity Supplement

 描述(申请人提供)：转录激活子、共激活子及其复合体的构象动力学支持受调控的转录。各个构象之间的低能垒意味着每个参与者可以使用同一组氨基酸来识别不同的结合伙伴，每个复合体具有不同的构象。假设每个构象都通过与其他结合伙伴的变构通信与独特的功能结果相关。然而，这个模型从来没有被测试过，主要是因为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。着眼于两个功能上重要的辅助激活基序，KIX(神经病理性疼痛，神经退行性疾病)和酸(癌症，病毒感染)，我们将开发共价化学辅助伴侣，它们单独稳定辅助激活物的特定构象，并与同源配体形成复合体。这些辅助伴侣分子将被用来严格表征处于不同构象和组装状态的辅助活化剂的结构(X射线结晶学、核磁共振波谱)和动力学(瞬时动力学、平衡结合、计算分析)。对体外数据的补充将是在细胞中进行化学辅助伴侣的实验，从而确定调控转录输出的相互作用和构象。通过这些研究，将确定这些疾病相关转录复合体的小分子靶向的变构结合部位。

 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.