USE OF THE THIOL-SPECIFIC DERIVATIZING AGENT N-IODOACETYL-3[I-125]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) c ontains five cysteine residues, with three of them being spaced close to one another in the steroid binding pocket, The HBD also contains th e contact region for the chaperone protein hsp90, which must be bound to the GR for it to have a steroid binding conformation, Binding of hs p90 to the receptor through its HBD inactivates the DNA binding domain (DBD), The DBD contains a number of cysteines essential to its DNA bi nding activity, Here, we assess the effects of hsp90 binding on the ac cessibility of cysteine residues in both the HBD and DBD to derivatiza tion by a thiol-specific reagent, We report that N-iodoacetyltyrosine (IAT) inactivates steroid binding activity of the immunopurified, untr ansformed GR hsp90 complex in a manner that is prevented by the sulfhy dryl reagents cysteine and dithiothreitol but is not reversed by them, The I-125-labeled IAT derivative N-iodoacetyl-3-[I-125]iodotyrosine ( [I-125]IAIT) covalently labels the immunopurified, hsp90-bound recepto r in a thiol-specific manner, Dissociation of hsp90 leads to an simila r to 2-fold increase in [I-125]IAIT labeling of the full-length, 100-k Da GR. The increase in thiol labeling is related to the presence of hs p90 because it is blocked by molybdate, which prevents hsp90 dissociat ion, Cleavage of the [I-125]IAIT-labeled receptor with trypsin yields a 15-kDa labeled fragment containing the DBD and a 30-kDa labeled frag ment 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 in crease in [I-125]IAIT labeling of the 15-kDa fragment and a 50% decrea se in labeling of the 30-kDa fragment, These data are consistent with the proposal that dissociation of hsp90 from the GR produces a conform ational change in the HBD such that some of the thiols that are expose d in the GR hsp90 complex become buried and are no longer accessible t o the [I-125]IAIT probe, In contrast, binding of the GR to hsp90 restr icts access of cysteines in the DBD to this small thiol-derivatizing a gent, a restriction that is relieved as a result of unmasking or confo rmational change accompanying hsp90 dissociation.