The assembly of progesterone receptor (PR) heterocomplexes in vitro involves at least eight components of the molecular chaperone machinery, and as earlier reports have shown, these proteins exhibit complex, dynamic, but ordered, interactions with one another and PR. Using the selective hsp90 binding agent geldanamycin (GA), we have found that PR assembly in vitro can be arrested at a previously observed intermediate assembly step. Like mature PR complexes, the intermediate complexes contain hsp90, but they differ from mature complexes by the presence of hsp70, p60, and p48 and the absence of immunophilins and p23. Arrest of PR assembly is likely due to GA's ability to directly block binding of p23 to hsp90. An important functional consequence of GA-mediated assembly arrest in vitro is the inability of the resulting PR complexes to bind progesterone, despite the presence of hsp90 in the receptor complexes. The biological significance of the in vitro observations is demonstrated by GA's ability to (i) rapidly block PR's hormone binding capacity in intact cells and (ii) alter the composition of COS cell PR complexes in a manner similar to that observed during in vitro reconstitutions. An updated model for the cyclic assembly pathway of PR complexes that incorporates the present findings with earlier results is presented.In the absence of hormone, steroid receptors are known to exist in heteromeric complexes containing multiple proteins, including heat shock protein 90 (hsp90) (for reviews see references 20 and 33). In order to understand the role of these proteins in regulating receptor assembly and hormone signalling, we and others have used a cell-free rabbit reticulocyte lysate (RL) system to support assembly of functional progesterone (30) and glucocorticoid (24) receptor complexes in vitro. For progesterone receptor (PR) assembly in vitro, at least eight distinct proteins have been identified as taking part in the ordered assembly of PR complexes (26,31). hsp70 can function as a molecular chaperone in promoting protein folding, and hsp90 may be able to promote folding as well (reviewed in reference 9). The other six proteins have not been shown to have individual chaperone activity, but each commonly associates with hsp70 and/or hsp90, and it is possible that each serves at least as an accessory in chaperone-mediated processes. Thus, all eight receptor-associated proteins can be considered components of the overall molecular chaperone machinery in eukaryotic cytoplasm.Importantly, proper assembly with hsp90 is required to establish and maintain PR's high-affinity progesterone binding state (26), as was previously demonstrated for glucocorticoid receptor (24). In vitro assembly of PR complexes competent for hormone binding requires ATP, Mg 2ϩ , K ϩ , and near-physiological temperature in addition to several protein factors (6-8, 12, 24, 26, 30, 31). A working model outlining the complex and dynamic interactions occurring during PR assembly in vitro was proposed earlier (26). In that model, a transient PR compl...

The ability to bind immunosuppressive drugs such as cyclosporin and FK506 defines the immunophilin family of proteins, and the FK506-binding proteins form the FKBP subfamily of immunophilins. Some FKBPs, notably FKBP12 (the 12-kDa FK506-binding protein), have defined roles in regulating ion channels or cell signaling, and well established structures. Other FKBPs, especially the larger ones, participate in important biological processes, but their exact roles and the structural bases for these roles are poorly defined. FKBP51 (the 51-kDa FKBP) associates with heat shock protein 90 (Hsp90) and appears in functionally mature steroid receptor complexes. In New World monkeys, FKBP51 has been implicated in cortisol resistance. We report here the x-ray structures of human FKBP51, to 2.7 Å, and squirrel monkey FKBP51, to 2.8 Å, by using multiwavelength anomalous dispersion phasing. FKBP51 is composed of three domains: two consecutive FKBP domains and a three-unit repeat of the TPR (tetratricopeptide repeat) domain. This structure of a multi-FKBP domain protein clarifies the arrangement of these domains and their possible interactions with other proteins. The two FKBP domains differ by an insertion in the second that affects the formation of the progesterone receptor complex.