Hsp90 is the most abundant molecular chaperone in the eukaryotic cell. One of the most stringent clients is the glucocorticoid receptor (GR), whose in vivo function strictly depends on the interaction with the Hsp90 machinery. However, the molecular mechanism of this interaction has been elusive. Here we have reconstituted the interaction of Hsp90 with hormone-bound GR using purified components. Our biochemical and structural analyses define the binding site for GR on Hsp90 and reveal that binding of GR modulates the conformational cycle of Hsp90. FRET experiments demonstrate that a partially closed form of the Hsp90 dimer is the preferred conformation for interaction. Consistent with this, the conformational cycle of Hsp90 is decelerated, and its ATPase activity decreases. Hsp90 cochaperones differentially affect formation of the Hsp90-GR complex, serving as control elements for cycle progression and revealing an intricate interplay of client and cochaperones as molecular modulators of the Hsp90 machine.

One function of the glucocorticoid receptor (GR) in corticotroph cells is to suppress the transcription of the gene encoding proopiomelanocortin (POMC), the precursor of the stress hormone adrenocorticotropin (ACTH). Cushing disease is a neuroendocrine condition caused by partially glucocorticoid-resistant corticotroph adenomas that excessively secrete ACTH, which leads to hypercortisolism. Mutations that impair GR function explain glucocorticoid resistance only in sporadic cases. However, the proper folding of GR depends on direct interactions with the chaperone heat shock protein 90 (HSP90, refs. 7,8). We show here that corticotroph adenomas overexpress HSP90 compared to the normal pituitary. N- and C-terminal HSP90 inhibitors act at different steps of the HSP90 catalytic cycle to regulate corticotroph cell proliferation and GR transcriptional activity. C-terminal inhibitors cause the release of mature GR from HSP90, which promotes its exit from the chaperone cycle and potentiates its transcriptional activity in a corticotroph cell line and in primary cultures of human corticotroph adenomas. In an allograft mouse model, the C-terminal HSP90 inhibitor silibinin showed anti-tumorigenic effects, partially reverted hormonal alterations, and alleviated symptoms of Cushing disease. These results suggest that the pathogenesis of Cushing disease caused by overexpression of heat shock proteins and consequently misregulated GR sensitivity may be overcome pharmacologically with an appropriate HSP90 inhibitor.

Allostery has been studied for many decades yet it remains challenging to determine experimentally how it occurs at a molecular level. We have developed an approach combining isothermal titration calorimetry, circular dichroism, and nuclear magnetic resonance spectroscopy to quantify allostery in terms of protein thermodynamics, structure, and dynamics. This strategy was applied to study the interaction between aminoglycoside N-(6′)-acetyltransferase-Ii and one of its substrates, acetyl coenzyme A. It was found that homotropic allostery between the two active sites of the homo-dimeric enzyme is modulated by opposing mechanisms. One follows a classical KNF paradigm while the other follows a recently-proposed mechanism in which partial unfolding of the subunits is coupled to ligand binding. Competition between folding, binding, and conformational changes represents a new way to govern energetic communication between binding sites. Allostery is a key feature of biological systems in which covalent modification or ligand binding at one site influences the activity at distant sites in a macromolecule or macromolecular assembly. Allosteric regulation plays a central role in metabolism and cell signaling, and has been identified as a source of new drug targets 1-5. Thus detailed descriptions of allostery have far reaching implications. A number of systems have been explained in terms of mechanistic models, yet allostery takes a variety of forms and many details of this phenomenon remain unresolved 6-9. Homotropic allostery (or homotropic cooperativity) involves interactions between a macromolecular system and two or more identical ligands. Cooperativity implies that binding depends on the ligation state, i.e. the first ligand is bound with a different affinity than the second, etc. This occurs frequently in oligomeric proteins and many theoretical models have focused on such systems. For Correspondence should be addressed to A.M.