STRUCTURAL STUDIES OF PAS DOMAIN SIGNALING MECHANISMS

描述（申请人描述）：PAS结构域介导蛋白/蛋白 调节广泛生物功能的蛋白质之间的相互作用， 包括基因表达。几种PAS的蛋白结合能力 结构域由小的有机配体调节，为这些结构域提供了一种机制。 化合物来调节复杂的生物活性。这些的错误调节 途径是有害的，正如PAS/毒素的中心作用所证明的那样， 复合物在发展几种环境的致癌作用 毒素拟议的研究将描述如何PAS域具体 与配体和其他蛋白质相互作用，以及这些结合事件是如何 整合在小的（100-120个残基）结构域内。为了实现这一点，基于NMR的 结构性研究主要包括三个方面：研究 单PAS结构域从四个真核生物蛋白质，使用新的方法， 快速鉴定这些结构域的可溶性片段用于NMR研究。结构 已经使用标准方法获得了关于两个这样的域的信息（12 和20 kDa），并表明配体和蛋白质结合位点是灵活的， 缺乏互动的伙伴。2）。PAS/配体复合物的研究。 从无配体结构域获得的信息开始，小分子 将通过基于NMR的方法筛选文库以鉴定潜在的配体。 这些研究的初步数据表明，几种化合物结合到 正在调查的PAS领域之一，僵化了几个灵活的 在无配体形式中观察到的区域。这些构象变化将是 通过检查PAS/配体的结构和动力学进行了更详细的研究 配合物3）。PAS/蛋白质复合物的研究。其中大部分是由 PAS/PAS异二聚化，促进使用同位素标记， 简化了这些大（约30 kDa）复合物的NMR谱。混合 将使用分辨率方法，通过对接生成复杂的模型 通过使用多个高分辨率结构的各个域， 在完整的复合体上获得了低分辨率的限制。PAS的这些观点 不同构象的结构域将提供对 它们的调节，并可能开辟新的方法来设计治疗 可以组装/分解PAS介导的复合物的试剂。

Description (applicant's description): PAS domains mediate protein/protein interactions among proteins that regulate a wide range of biological functions, including gene expression. The protein-binding capabilities of several PAS domains are modulated by small organic ligands, providing a mechanism for these compounds to regulate complex biological activities. Misregulation of these pathways is deleterious, as demonstrated by the central role of PAS/toxin complexes in developing the carcinogenic effects of several environmental toxins. The proposed research will characterize how PAS domains specifically interact with ligands and other proteins and how these binding events are integrated within a small (100-120 residue) domain. To achieve this, NMR-based structural studies will be conducted in three general areas: 1). Studies of single PAS domains from four eukaryotic proteins, using novel methods to rapidly identify soluble fragments of these domains for NMR studies. Structural information has been obtained using standard methods on two such domains (12 and 20kDa) and suggests that ligand- and protein-binding sites are flexible in the absence of interacting partners. 2). Studies of PAS/ligand complexes. Starting with the information obtained on ligand-free domains, small molecule libraries will be screened by NMR-based methods to identify potential ligands. Preliminary data from these studies demonstrate that several compounds bind to one of the PAS domains under investigation, rigidifying several flexible regions observed in the ligand-free form. These conformational changes will be studied in more detail by examining the structures and dynamics of PAS/ligand complexes. 3). Studies of PAS/protein complexes. Most of these are formed by PAS/PAS heterodimerization, facilitating the use of isotopic labeling to simplify the NMR spectra of these large (about 30kDa) complexes. A mixed resolution approach will be used, generating models of the complex by docking by high resolution structures of the individual domains using multiple low-resolution restraints acquired on the intact complex. These views of PAS domains in different conformations will provide insight into the mechanism of their regulation and possibly open new approaches to the design of therapeutic agents that could assemble/disassemble PAS-mediated complexes.