NMR Studies of Chaperone-Client Protein Interactions

描述(申请人提供)：在真核生物中，伴侣Hsp90及其辅助伴侣与一些至关重要的细胞因子相互作用并稳定下来，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。尽管最近发表了一些关于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，即使形成了晶体，如果这些络合物实际上具有显著的灵活性，那么从它们衍生的结构可能会产生误导。我们提出了一个核磁共振波谱的方法来解决这个问题，以提供有关这些络合物在溶液中的信息。有些蛋白质组分比传统的溶液核磁共振研究的要大，但我们已经设计了实验，使我们能够使用本实验室最近开发的分析氢/氢交换的方法，对Hsp90-客户相互作用的性质得出有效的结论。这项强大的新技术使用了非质子溶剂DMSO来捕获标记物种，消除了氚标记的混乱，将用于探索客户蛋白和辅助伴侣与Hsp90及其结构域的结构、稳定性和相互作用。对所提出的实验方法的初步验证将检查两个Hsp90辅助伴侣p23和Aha1的相互作用位点。掩埋在络合物形成上的表面将通过比较15N标记的共伴侣在存在和不存在伴侣的情况下对酰胺质子的氢交换保护来检测。随后，优化的方法将被应用于Hsp90与客户蛋白的相互作用，包括肿瘤抑制基因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，低氧诱导因子-2α的PaSB域，以及囊性纤维化跨膜受体的核苷酸结合域。Hsp90本身的结合部位和构象变化将通过甲基特异性标记和高分辨率核磁共振进一步表征。在过去的几年里，随着人们认识到Hsp90在细胞过程中的核心作用，人们对Hsp90系统的兴趣呈指数级增长。然而，对溶液中的伴侣-客户络合物的结构了解仍然很少。这项提议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Hsp90参与了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，以及帮助稳定和跨膜运输蛋白质。HSP90抑制剂是制药行业密集研究的主题，作为癌症的一般疗法，HSP90及其辅助伴侣似乎也与导致囊性纤维化等疾病的蛋白质有关。我们的研究将阐明这些络合物在溶液中的结构和动力学性质，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.