Use of the thiol-specific derivatizing agent N-iodoacetyl-3-[125I]iodotyrosine to demonstrate conformational differences between the unbound and hsp90-bound glucocorticoid receptor hormone binding domain.
The hormone binding domain (HBD) of the glucocorticoid receptor (GR) contains five cysteine residues, with three of them being spaced close to one another in the steroid binding pocket. The HBD also contains the contact region for the chaperone protein hsp90, which must be bound to the GR for it to have a steroid binding conformation. Binding of hsp90 to the receptor through its HBD inactivates the DNA binding domain (DBD). The DBD contains a number of cysteines essential to its DNA binding activity. Here, we assess the effects of hsp90 binding on the accessibility of cysteine residues in both the HBD and DBD to derivatization by a thiol-specific reagent. We report that N-iodoacetyltyrosine (IAT) inactivates steroid binding activity of the immunopurified, untransformed GR.hsp90 complex in a manner that is prevented by the sulfhydryl reagents cysteine and dithiothreitol but is not reversed by them. The 125I-labeled IAT derivative N-iodoacetyl-3-[125I]iodotyrosine ([125I]IAIT) covalently labels the immunopurified, hsp90-bound receptor in a thiol-specific manner. Dissociation of hsp90 leads to an approximately 2-fold increase in [125I]IAIT labeling of the full-length, 100-kDa GR. The increase in thiol labeling is related to the presence of hsp90 because it is blocked by molybdate, which prevents hsp90 dissociation. Cleavage of the [125I]IAIT-labeled receptor with trypsin yields a 15-kDa labeled fragment containing the DBD and a 30-kDa labeled fragment containing all of the cysteines in the HBD and the contact region for hsp90. Dissociation of hsp90 from the GR results in a 2.3-fold increase in [125I]IAIT labeling of the 15-kDa fragment and a 50% decrease in labeling of the 30-kDa fragment. These data are consistent with the proposal that dissociation of hsp90 from the GR produces a conformational change in the HBD such that some of the thiols that are exposed in the GR*hsp90 complex become buried and are no longer accessible to the [125I]IAIT probe. In contrast, binding of the GR to hsp90 restricts access of cysteines in the DBD to this small thiol-derivatizing agent, a restriction that is relieved as a result of unmasking or conformational change accompanying hsp90 dissociation.
Stancato
L F
LF
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
Silverstein
A M
AM
Gitler
C
C
Groner
B
B
Pratt
W B
WB
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry; D007739:L Cells; D046911:Macromolecular Substances; D051379:Mice; D007470:Monoiodotyrosine / Q000031:analogs & derivatives / Q000737:chemistry; D010449:Peptide Mapping; D011487:Protein Conformation; D011965:Receptors, Glucocorticoid / Q000737:chemistry*; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents / Q000737:chemistry
D018841:HSP90 Heat-Shock Proteins; D046911:Macromolecular Substances; D011965:Receptors, Glucocorticoid; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; C087944:N-iodoacetyl-3-iodotyrosine; D007470:Monoiodotyrosine