Glucocorticoid receptor complexes form cooperatively with the Hsp90 co-chaperones Pp5 and FKBPs

Abstract

The function of steroid receptors in the cell depends on the chaperone machinery of Hsp90, as Hsp90 primes steroid receptors for hormone binding and transcriptional activation. Several conserved proteins are known to additionally participate in receptor chaperone assemblies, but the regulation of the process is not understood in detail. Also, it is unknown to what extent the contribution of these cofactors is conserved in other eukaryotes. We here examine the reconstituted C. elegans and human chaperone assemblies. We find that the nematode phosphatase PPH-5 and the prolyl isomerase FKB-6 facilitate the formation of glucocorticoid receptor (GR) complexes with Hsp90. Within these complexes, Hsp90 can perform its closing reaction more efficiently. By combining chemical crosslinking and mass spectrometry, we define contact sites within these assemblies. Compared to the nematode Hsp90 system, the human system shows less cooperative client interaction and a stricter requirement for the co-chaperone p23 to complete the closing reaction of GR•Hsp90•Pp5/Fkbp51/ Fkbp52 complexes. In both systems, hormone binding to GR is accelerated by Hsp90 alone and in the presence of its cofactors. our results show that cooperative complex formation and hormone binding patterns are, in many aspects, conserved between the nematode and human systems. Hsp90 is an ATP-driven molecular machine, highly abundant in the cytosol. It is recruited to client proteins that require energy-intense rearrangements and supports these reactions by performing nucleotide-induced conformational changes 1,2. These changes lead to the compaction of the Hsp90 dimer and the transient formation of a ring-like structure, where the N-terminal domains are dimerized in addition to the chaperone's permanent C-terminal dimerization 3. Only in the bacterial Hsp90 system are these changes performed and controlled by Hsp90 itself. In the eukaryotic systems, client-specific cofactors are present, which tailor the Hsp90 machinery into a client-specific mode and regulate the conformational changes while providing further interaction sites for the clients 4. This has been investigated for the Hsp90-dependent maturation of protein kinases with the help of the cofactor Cdc37 and for the chaperoning of steroid receptors, where a larger set of cofactors participates 5-7. Partly, these reactions have been reconstituted with recombinant proteins to identify interaction mechanisms, but mostly addressing the cofactors' interaction with Hsp90 in the absence of clients. Hsp90-containing protein complexes were first identified in the 1980s 8,9. In these studies, steroid hormone receptors, such as the glucocorticoid, mineralocorticoid or progesterone receptors, were isolated from vertebrate cells and the associated proteins were identified and studied by western blot analyses. Hsp90 and several other proteins were detected in these complexes, leading to the identification of Fkbp51, Fkbp52, Cyp40, Hop, p23, Hip and Pp5 as components of steroid hormone receptor complexes 10-14. The gluco...

Glucocorticoid resistance conferring mutation in the C-terminus of GR alters the receptor conformational dynamics

“…SHR crystal structures with agonist ligands like DEX, have shown that the C-terminal α-helix, the activation function-2 (AF-2), serves in forming the lid of the ligand binding pocket 35 , 36 . Given that the wild type GR protein is rather unstable, we employ a GR variant, GRm, which is stabilized by mutagenesis and has previously been used to shed light on the GR•Hsp90 interaction 23 – 25 . To study the influence of the point mutation, we utilize protein constructs that contain the DBD, hinge region, and LBD of GR.…”

“…Moreover, the ligand-activated, membrane-bound GR is reported to mediate non-genomic events, such as triggering the activation of kinase signaling cascades, including the mitogen.-activated protein kinase (MAPK) or the phosphatidylinositol 3-kinase (PI3K) pathways 22 . Even though much progress has been done on comprehending the Hsp90-client interplay, with steroid receptors playing a key role in such studies, as obligate Hsp90 clients, the molecular mechanism of this interaction is sti l l enigmatic and the actual transformations on GR are yet to be clarified 14 , 23 – 25 .…”

Cortisol promotes stress tolerance via DAF-16 in Caenorhabditis elegans

“…Although a previous study suggested that invertebrates, including C. elegans , do not possess GR, it is possible that cortisol affected C. elegans via another kind of nuclear receptors. A recent study reported that among HSP-90-client nuclear receptors, such as human GR, the ligand-binding domains of NHR-25, NHR-47, DAF-12, and FAX-1 have high homology with human GR [ 30 ], suggesting that there are nuclear receptors and signaling pathways functionally similar to GR in nematodes. Thus, the corresponding receptor and the pathways that involve DAF-16 could be identified in future studies.…”

HSP-90/kinase complexes are stabilized by the large PPIase FKB-6

“…dF/dt-plots from one rotor containing 14 samples were calculated by using the optimal time range of the experiment, subtracting the respective scans from each other and averaging over several such differentials with the in-house script diffUZ. The obtained averages were normalized against the plateau value of the fluorescence as described 15 . Plots were then fit to bi-Gaussian functions to get good estimates of the sedimentation coefficient s 20,w and peak amplitudes.…”