Folding and stability of the ligand‐binding domain of the glucocorticoid receptor

A complex pathway involving many molecular chaperones has been proposed for the folding, assembly, and maintenance of a high‐affinity ligand‐binding form of steroid receptors in vivo, including the glucocorticoid receptor. To better understand this intricate folding and assembly process, we studied the folding of the ligand‐binding domain of the glucocorticoid receptor in vitro. We found that this domain can be refolded into a compact, highly structured state in vitro in the absence of chaperones. However, the presence of zwitterionic detergent is required to maintain the domain in a soluble form. In this state, the protein is dimeric and has considerable helical structure as shown by far‐UV circular dichroism. Further investigation of the properties of this in vitro refolded state show that it is stable and resistant to denaturation by heat or low concentrations of chemical denaturants. A detailed analysis of the unfolding equilibria using three different structural probes demonstrated that this state unfolds via a highly populated dimeric intermediate state. Together, these data clearly show that the ligand‐binding domain of the glucocorticoid receptor does not require chaperones for folding per se. However, this in vitro refolded state binds the ligand dexamethasone only weakly (K_d = 45 μM) compared to the in vivo assembled receptor (K_d = 3.4 nM). We suggest that the role of Hsp90 and associated chaperones is to bind to, and stabilize, a specific conformational state of the receptor which binds ligand with high affinity.