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Solution structures and conformational dynamics of the molecular chaperone Hsp90


The molecular chaperone Hsp90 is an essential eukaryotic protein making up 1-2% of all cytosolic proteins. Hsp90 is vital for the maturation and maintenance of a wide variety of substrate proteins largely involved in signaling and regulatory processes. Many of these substrates have been implicated in cancer and other diseases making Hsp90 an attractive target for therapeutics. Hsp90 depends upon its intrinsic ATPase activity for function. Crystal structures of the bacterial Hsp90 homolog, HtpG, and the yeast Hsp90 homolog reveal large domain rearrangements between the nucleotide-free and the nucleotide-bound forms. Using small angle X-ray scattering and newly developed molecular modeling techniques, I investigated the solution states of HtpG. In solution, nucleotide-free HtpG adopts a more extended conformation than observed in the crystal, and upon the addition of AMPPNP, HtpG is in equilibrium between this open state and a closed state that is in good agreement with the yeast AMPPNP crystal structure. For apo HtpG, I also identified a two-state pH-dependent conformational equilibrium between the open, extended state and a U-shaped state resembling the crystal structure of the ER homolog Grp94. Through mutagenesis I successfully modulated the conformational equilibrium at neutral pH and showed that the conformations are functionally distinct in the context of a citrate synthase aggregation assay. These studies provide a unique view of HtpG conformational dynamics and a new model for the role of nucleotide in effecting conformational change. They also provide the first linkage between a specific conformation and chaperone function. The conformations of the eukaryotic homologs have been less well characterized, especially in the absence of nucleotide. To better define the similarities and differences between the prokaryotic and eukaryotic proteins, I studied the solution states of cytosolic yeast and human Hsp90 as well as the mouse endoplasmic reticulum homolog, Grp94. I showed that all three proteins adopt an extended, chair-like conformation distinct from the extended conformation observed for the bacterial Hsp90. Ultimately Hsp90 must be studied in the context of client proteins. To this end, I worked to develop in vitro assays for the Hsp90-dependent activation of the tyrosine kinase c-src.

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