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.
The chaperone Hsp90 is involved in many critically important cellular functions, including stabilizing the structures of client proteins such as transcription factors and kinases, and assisting in the movement of proteins across membranes. We still do not know the structural basis for the interaction between Hsp90 and its clients. This proposal aims to use new methods to prepare these complexes, avoiding the difficulties posed by the instability of the client proteins, and to utilize a number of state-of-the-art biophysical methodsto obtain new insights into their structure.
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