NMR Studies of Chaperone-Client Protein Interactions

描述（由申请人提供）：在真核生物中，伴侣蛋白Hsp90及其共伴侣蛋白与许多至关重要的细胞因子相互作用并稳定它们，包括类固醇受体、激酶和转录因子。该项目将研究伴侣蛋白与这些“客户”蛋白的相互作用。尽管最近发表了许多关于Hsp90的x射线研究，但Hsp90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，而且即使形成了晶体，如果这些配合物实际上具有显著的柔韧性，那么由此产生的结构可能会产生误导。我们提出了一种核磁共振光谱方法来解决这个问题，以提供这些复合物在溶液中的信息。一些蛋白质成分比传统的溶液核磁共振研究更大，但实验已经设计好，将使我们能够得出关于hsp90客户端相互作用性质的有效结论，使用该实验室最近开发的氢/氘交换分析方法。该技术利用非质子溶剂DMSO捕获被标记的物质，消除氘标记的混乱，将用于探索客户蛋白和共伴侣蛋白与Hsp90及其结构域的结构、稳定性和相互作用。所提出的实验方法的初步验证将检查两个Hsp90共同伴侣p23和Aha1的相互作用位点。通过比较15n标记的共伴侣在存在和不存在伴侣的情况下酰胺质子的氘交换保护，可以检测复合物形成后埋藏的表面。优化后的方法将应用于Hsp90与客户蛋白的相互作用，包括肿瘤抑制因子p53的核心dna结合域、糖皮质激素受体和雌激素受体的配体结合域、缺氧诱导因子- 2α的PASB结构域和囊性纤维化跨膜受体的核苷酸结合域。Hsp90本身的结合位点和构象变化将通过甲基特异性标记和高分辨率NMR进一步表征。在过去的几年中，随着Hsp90在细胞过程中的核心作用被认识到，对Hsp90系统的兴趣呈指数增长。然而，对溶液中伴侣-客户复合物的结构理解仍然很少。本提案旨在使用最先进的核磁共振方法来解决这个问题。公共卫生相关性：伴侣蛋白Hsp90参与至关重要的细胞功能，包括稳定转录因子和激酶等客户蛋白的结构，并帮助蛋白质的稳定和跨膜运输。Hsp90抑制剂是制药行业研究的重点，作为癌症的一般疗法，Hsp90及其共同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态性质，为未来的药物设计工作提供指导。

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.