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 microM) 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.
McLaughlin
Stephen H
SH
Cambridge University Chemical Laboratory, UK.
Jackson
Sophie E
SE
D002850:Chromatography, Gel; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism; D019791:Guanidine / Q000494:pharmacology; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011965:Receptors, Glucocorticoid / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D013050:Spectrometry, Fluorescence
D018841:HSP90 Heat-Shock Proteins; D008024:Ligands; D011965:Receptors, Glucocorticoid; D011994:Recombinant Proteins; D019791:Guanidine