Interactions between Hsp90, Co-chaperones and Client Proteins

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，所述细胞因子包括类固醇激素受体、细胞激酶和转录因子。虽然我们对分子伴侣和共分子伴侣的结构已经有了很大的了解，但Hsp 90与其客户之间相互作用的结构基础仍然是未知的。根本的问题是，我们仍然不了解客户蛋白质与Hsp 90分子伴侣结合时的物理状态，我们对客户蛋白质在Hsp 90分子上的接触位置只有一个粗略的想法。在这些系统上获得数据的部分困难在于客户蛋白质通常极难以适合于结构分析的量制备。毫无疑问，这是细胞在其稳定过程中使用Hsp 90的主要动机。我们提出了创新的客户端蛋白-热休克蛋白90复合物的制备方法，通过添加其他必要的成分的伴侣级联的真核细胞和重新思考的可能形式的高亲和力的客户端蛋白。我们的第一个目标将是表征无锌形式的转录因子p53和全长热休克蛋白90二聚体之间的相互作用，使用一组结构测量，可以询问这样一个大的复杂。这些测量包括小角X射线散射、室温EPR、荧光方法和电子显微镜。第二个具体目标将涉及共同监护人对系统的影响。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。NMR光谱和其他方法将被用来表征这种二元相互作用，以及我们观察到的三元相互作用与热休克蛋白90的添加。在第三个具体的目标，我们将研究糖皮质激素受体配体结合域（GR-LBD）和热休克蛋白90的复合物，适应的方法，已在文献中使用，以证明这种相互作用的存在下，在哺乳动物细胞提取物。该复合物的新制备方法包括组氨酸标记的GR-LBD蛋白的过表达以及在复合物的制备中掺入来自伴侣级联的辅助蛋白。拟议工作的成功结束不仅将为伴侣-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其细胞环境中采样的结构状态范围提供重要的新见解：我们只是最近才开始认识到，除了“完全和稳定折叠”的蛋白质构象状态，由传统结构研究代表的状态可以在细胞中存在并完全起作用。

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.