Interactions between Hsp90, Co-chaperones and Client Proteins

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，包括类固醇激素受体、细胞激酶和转录因子。虽然人们对伴侣和辅助伴侣的结构已经有了很多了解，但Hsp90与其客户之间相互作用的结构基础仍不清楚。根本问题是，当客户蛋白与Hsp90分子伴侣结合时，我们仍然不了解客户蛋白的物理状态，而且我们对客户蛋白在Hsp90分子上的哪里接触只有一个粗略的了解。在这些系统上获得数据的困难之一是，客户蛋白通常极难制备出适合结构分析的数量。毫无疑问，这是细胞采用Hsp90来稳定其稳定性的主要动机。我们提出了制备客户蛋白-Hsp90复合体的创新方法，方法是添加真核细胞伴侣级联的其他必要组件，并重新考虑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可以询问如此大的复合体的结构测量来表征无锌形式的转录因子p53和全长Hsp90二聚体之间的相互作用。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制度的影响。初步数据显示，在p53 DBD客户蛋白和p23辅助伴侣之间存在可测量的相互作用。核磁共振波谱和其他方法将被用来表征这种二元相互作用，以及我们观察到的添加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的复合体，采用文献中已经使用的方法来证明在哺乳动物细胞提取物中存在这种相互作用。一种新的复合体的制备方法包括过表达组氨酸标记的GR-LBD蛋白以及在复合体的制备过程中掺入来自伴侣级联的辅助蛋白。这项拟议工作的成功结束不仅将为伴侣-客户蛋白质复合体的具体性质提供重要的新见解，而且将为蛋白质在其细胞环境中采样的结构状态的范围提供重要的新见解：我们直到最近才开始认识到，除了传统结构研究中典型的“完全和稳定折叠”状态外，蛋白质构象状态可能存在于细胞中并具有完全功能。

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.