NMR Studies of Chaperone-Client Protein Interactions

描述（由申请人提供）：在真核生物中，分子伴侣Hsp 90及其辅助分子伴侣与许多至关重要的细胞因子相互作用并使其稳定，这些细胞因子包括类固醇受体、激酶和转录因子。这个项目将研究分子伴侣与这些“客户”蛋白质的相互作用。尽管最近发表了一些X射线研究的热休克蛋白90，热休克蛋白90结合的客户蛋白的结构状态是完全未知的。这样的体系很难结晶，即使形成了晶体，如果这些络合物实际上具有显著的柔性，那么由它们衍生的结构可能会产生误导。我们提出了一个NMR光谱的方法来解决这个问题，这些配合物在解决方案中提供信息。一些蛋白质组分比传统上通过溶液NMR研究的要大，但实验的设计将使我们能够使用本实验室最近开发的用于分析氢/氘交换的方法，对Hsp 90-客户端相互作用的性质得出有效的结论。这种强大的新技术，它采用非质子溶剂DMSO捕获标记的物种和消除混乱的氘标记，将被用来探测客户蛋白和辅助分子伴侣的结构，稳定性和与热休克蛋白90及其结构域的相互作用。所提出的实验方法的初步验证将检查两个热休克蛋白90共分子伴侣，p23和Aha 1的相互作用位点。通过比较存在和不存在伴侣的情况下15 N标记的共伴侣样品中酰胺质子的氘交换保护，检测复合物形成后掩埋的表面。优化的方法将随后应用于Hsp 90与客户蛋白的相互作用，包括肿瘤抑制因子p53的核心DNA结合结构域，糖皮质激素受体和雌激素受体的配体结合结构域，缺氧诱导因子-2 α的PASB结构域，以及囊性纤维化跨膜受体的核苷酸结合结构域。结合位点和热休克蛋白90本身的构象变化将进一步通过甲基特异性标记和高分辨率NMR的特点。在过去的几年中，随着人们认识到Hsp 90在细胞过程中的核心作用，对Hsp 90系统的兴趣呈指数增长。尽管如此，仍然有很少的结构理解伴侣客户端复合物的解决方案。该提案旨在使用最先进的NMR方法来解决这个问题。公共卫生相关性：伴侣蛋白Hsp 90参与至关重要的细胞功能，包括稳定客户蛋白质（如转录因子和激酶）的结构，并帮助蛋白质的稳定和跨膜转运。Hsp 90抑制剂是制药行业中密集研究的主题，作为癌症的一般疗法，Hsp 90及其辅助分子伴侣似乎也与引起囊性纤维化等疾病的蛋白质有关。我们的研究将阐明这些复合物在溶液中的结构和动力学性质，作为未来药物设计工作的指导。

DESCRIPTION (provided by applicant): In eukaryotes, the chaperone Hsp90 and its co-chaperones interact with and stabilize a number of crucially important cellular factors, including steroid receptors, kinases and transcription factors. This project will study the interactions of chaperones with these "client" proteins. Despite the recent publication of a number of X-ray studies of Hsp90, the structural states of Hsp90-bound client proteins are completely unknown. Such systems are difficult to crystallize, and even if crystals are formed, structures derived from them may be misleading if significant flexibility is in fact characteristic of these complexes. We propose an NMR spectroscopic approach to this problem, to provide information on these complexes in solution. Some of the protein components are larger than have traditionally been studied by solution NMR, but experiments have been designed that will allow us to draw valid conclusions on the nature of Hsp90-client interactions, using methods recently developed in this laboratory for the analysis of hydrogen/deuterium exchange. This powerful new technique, which employs the aprotic solvent DMSO to trap the labeled species and eliminate scrambling of deuterium label, will be used to probe the structure, stability and interactions of client proteins and co-chaperones with Hsp90 and its domains. Initial validation of the proposed experimental methods will examine the interaction sites of two Hsp90 co-chaperones, p23 and Aha1. Surfaces buried upon complex formation will be detected by comparison of the deuterium-exchange protection of amide protons in samples of 15N-labeled co-chaperone in the presence and absence of chaperone. The optimized methodology will subsequently be applied to the interactions of Hsp90 with client proteins, including the core DNA-binding domain of the tumor suppressor p53, the ligand binding domains of glucocorticoid receptor and estrogen receptor, the PASB domain of hypoxia-inducible factor-2alpha, and the nucleotide binding domain of the cystic fibrosis transmembrane receptor. Binding sites and conformational changes in Hsp90 itself will be further characterized by methyl-specific labeling and high-resolution NMR. During the last few years, interest in the Hsp90 system has increased exponentially, as the central role of Hsp90 in cellular processes is recognized. Nevertheless, there is still very little structural understanding of chaperone-client complexes in solution. This proposal aims to use state-of-the-art NMR methods to address this problem. PUBLIC HEALTH RELEVANCE: The chaperone Hsp90 is involved in critically important cellular functions, including stabilizing the structures of client proteins such as transcription factors and kinases, and aiding in the stabilization and transport of proteins across membranes. Hsp90 inhibitors are the subject of intense study in the pharmaceutical industry, as general therapies for cancers, and Hsp90 and its co-chaperones also appear to be involved with proteins that give rise to diseases such as cystic fibrosis. Our studies will elucidate the structural and dynamic nature of these complexes in solution, as a guide for future drug design efforts.