Interactions between Hsp90, Co-chaperones and Client Proteins

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，包括类固醇激素受体、细胞激酶和转录因子。尽管人们对伴侣蛋白和共伴侣蛋白的结构了解甚多，但Hsp90与其客户端相互作用的结构基础仍然未知。最根本的问题是，当客户蛋白与Hsp90伴侣蛋白结合时，我们仍然不了解客户蛋白的物理状态，对于客户蛋白在Hsp90分子上的接触位置，我们只有一个粗略的概念。获得这些系统数据的部分困难在于客户蛋白通常极难制备出适合结构分析的数量。毫无疑问，这是细胞使用Hsp90来稳定它们的主要动机。我们提出了制备客户蛋白- hsp90复合物的创新方法，通过添加真核细胞的伴侣蛋白级联的其他必要成分，并通过重新思考高亲和力客户蛋白的可能形式。我们的第一个目标将是利用一组结构测量来表征转录因子p53的无锌形式和全长Hsp90二聚体之间的相互作用，这些测量可以询问如此大的复合体。这些测量方法包括小角x射线散射、室温EPR、荧光法和电子显微镜。第二个具体目标将涉及共同伴侣对该制度的影响。初步数据显示，在p53 DBD客户蛋白和p23共伴侣蛋白之间存在可测量的相互作用。核磁共振波谱和其他方法将用于表征这种二元相互作用，以及我们观察到的加入Hsp90的三元相互作用。在第三个特定目标中，我们将研究糖皮质激素受体配体结合域（GR-LBD）和Hsp90的复合物，采用文献中用于证明哺乳动物细胞提取物中存在这种相互作用的方法。一种新的复合物制备方法包括过表达组氨酸标记的GR-LBD蛋白，并在复合物制备过程中加入伴侣级联的辅助蛋白。这项工作的成功结论不仅将为伴侣-客户蛋白复合物的特定性质提供重要的新见解，而且还将为蛋白质在细胞环境中采样的结构状态范围提供重要的新见解：我们最近才开始认识到，传统结构研究中典型的“完全稳定折叠”状态之外的蛋白质构象状态可能存在并在细胞中充分发挥作用。

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 hormone receptors, cellular kinases and transcription factors. Although a great deal is known about chaperone and co-chaperone structure, the structural basis for the interaction between Hsp90 and its clients remains unknown. The fundamental problem is that we still do not understand the physical state of the client protein when it is bound to the Hsp90 chaperone, and we have only a rough idea of where on the Hsp90 molecule the client protein makes contact. Part of the difficulty with obtaining data on these systems is that the client proteins ar generally extremely difficult to prepare in quantities suitable for structural analysis. Doubtless this is a major incentive for the cell to employ Hsp90 in their stabilization. We propose innovative methods of preparation of client protein-Hsp90 complexes, by addition of other necessary components of the chaperone cascade of the eukaryotic cell and by re-thinking the likely form of a high-affinity client protein. Our first aim will be to characterize interactions between a zinc-free form of the transcription factor p53 and the full-length Hsp90 dimer, using a set of structural measurements that can interrogate such a large complex. These measurements include small-angle X-ray scattering, room- temperature EPR, fluorescence methods and electron microscopy. The second specific aim will be concerned with the effect of co-chaperones on the system. Preliminary data show that there is a measurable interaction between the p53 DBD client protein and the p23 co-chaperone. NMR spectroscopy and other methods will be used to characterize this binary interaction, as well as the ternary interaction that we observe with the addition of Hsp90. In the third specific aim, we will examine the complex of the glucocorticoid receptor ligand-binding domain (GR-LBD) and Hsp90, adapting methods that have been used in the literature to demonstrate the presence of this interaction in mammalian cell extracts. A new preparation method for the complex includes the over-expression of a histidine-tagged GR-LBD protein together with the incorporation of accessory proteins from the chaperone cascade in the preparation of the complex. The successful conclusion of the proposed work will give important new insights not only into the specific nature of the chaperone-client protein complex, but also into the range of structural states sampled by proteins in their cellular environments: we have only recently begun to appreciate that protein conformational states other than the "fully and stably folded" states typified by traditional structural studies may be present and fully functional in the cell.