Structure of Hsp90-p23-GR reveals the Hsp90 client-remodelling mechanism.

Hsp90 is a conserved and essential molecular chaperone responsible for the folding and activation of hundreds of 'client' proteins1-3. The glucocorticoid receptor (GR) is a model client that constantly depends on Hsp90 for activity4-9. GR ligand binding was previously shown to nr inhibited by Hsp70 and restored by Hsp90, aided by the co-chaperone p2310. However, a molecular understanding of the chaperone-mediated remodelling that occurs between the inactive Hsp70-Hsp90 'client-loading complex' and an activated Hsp90-p23 'client-maturation complex' is lacking for any client, including GR. Here we present a cryo-electron microscopy (cryo-EM) structure of the human GR-maturation complex (GR-Hsp90-p23), revealing that the GR ligand-binding domain is restored to a folded, ligand-bound conformation, while being simultaneously threaded through the Hsp90 lumen. In addition, p23 directly stabilizes native GR using a C-terminal helix, resulting in enhanced ligand binding. This structure of a client bound to Hsp90 in a native conformation contrasts sharply with the unfolded kinase-Hsp90 structure11. Thus, aided by direct co-chaperone-client interactions, Hsp90 can directly dictate client-specific folding outcomes. Together with the GR-loading complex structure12, we present the molecular mechanism of chaperone-mediated GR remodelling, establishing the first, to our knowledge, complete chaperone cycle for any Hsp90 client.

Structural basis for glucocorticoid receptor recognition of both unmodified and methylated binding sites, precursors of a modern recognition element.

The most common form of DNA methylation involves the addition of a methyl group to a cytosine base in the context of a cytosine-phosphate-guanine (CpG) dinucleotide. Genomes from more primitive organisms are more abundant in CpG sites that, through the process of methylation, deamination and subsequent mutation to thymine-phosphate-guanine (TpG) sites, can produce new transcription factor binding sites. Here, we examined the evolutionary history of the over 36 000 glucocorticoid receptor (GR) consensus binding motifs in the human genome and identified a subset of them in regulatory regions that arose via a deamination and subsequent mutation event. GR can bind to both unmodified and methylated pre-GR binding sequences (GBSs) that contain a CpG site. Our structural analyses show that CpG methylation in a pre-GBS generates a favorable interaction with Arg447 mimicking that made with a TpG in a GBS. This methyl-specific recognition arose 420 million years ago and was conserved during the evolution of GR and likely helps fix the methylation on the relevant cytosines. Our study provides the first genetic, biochemical and structural evidence of high-affinity binding for the likely evolutionary precursor of extant TpG-containing GBS.

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

The glucocorticoid receptor is a key regulator of essential physiological processes, which under the control of the Hsp90 chaperone machinery, binds to steroid hormones and steroid-like molecules and in a rather complicated and elusive response, regulates a set of glucocorticoid responsive genes. We here examine a human glucocorticoid receptor variant, harboring a point mutation in the last C-terminal residues, L773P, that was associated to Primary Generalized Glucocorticoid Resistance, a condition originating from decreased affinity to hormone, impairing one or multiple aspects of GR action. Using in vitro and in silico methods, we assign the conformational consequences of this mutation to particular GR elements and report on the altered receptor properties regarding its binding to dexamethasone, a NCOA-2 coactivator-derived peptide, DNA, and importantly, its interaction with the chaperone machinery of Hsp90.

Single-molecule force spectroscopy reveals folding steps associated with hormone binding and activation of the glucocorticoid receptor.

The glucocorticoid receptor (GR) is a prominent nuclear receptor linked to a variety of diseases and an important drug target. Binding of hormone to its ligand binding domain (GR-LBD) is the key activation step to induce signaling. This process is tightly regulated by the molecular chaperones Hsp70 and Hsp90 in vivo. Despite its importance, little is known about GR-LBD folding, the ligand binding pathway, or the requirement for chaperone regulation. In this study, we have used single-molecule force spectroscopy by optical tweezers to unravel the dynamics of the complete pathway of folding and hormone binding of GR-LBD. We identified a "lid" structure whose opening and closing is tightly coupled to hormone binding. This lid is located at the N terminus without direct contacts to the hormone. Under mechanical load, apo-GR-LBD folds stably and readily without the need of chaperones with a folding free energy of [Formula: see text] The folding pathway is largely independent of the presence of hormone. Hormone binds only in the last step and lid closure adds an additional [Formula: see text] of free energy, drastically increasing the affinity. However, mechanical double-jump experiments reveal that, at zero force, GR-LBD folding is severely hampered by misfolding, slowing it to less than 1·s-1 From the force dependence of the folding rates, we conclude that the misfolding occurs late in the folding pathway. These features are important cornerstones for understanding GR activation and its tight regulation by chaperones.

The first crystal structure of a DNA-free nuclear receptor DNA binding domain sheds light on DNA-driven allostery in the glucocorticoid receptor.

The glucocorticoid receptor (GR) is a steroid hormone receptor of the nuclear receptor family that regulates gene expression in response to glucocorticoid hormone signaling. Interaction with specific GR DNA binding sequences causes conformational changes in the GR DNA binding domain (DBD) that result in recruitment of specific sets of co-regulators that determine transcriptional outcomes. We have solved the crystal structure of GR DBD in its DNA-free state, the first such crystal structure from any nuclear receptor. In contrast to previous NMR structures, this crystal structure reveals that free GR DBD adopts a conformation very similar to DNA-bound states. The lever arm region is the most variable element in the free GR DBD. Molecular dynamics of the free GR DBD as well as GR DBD bound to activating and repressive DNA elements confirm lever arm flexibility in all functional states. Cluster analysis of lever arm conformations during simulations shows that DNA binding and dimerization cause a reduction in the number of conformations sampled by the lever arm. These results reveal that DNA binding and dimerization drive conformational selection in the GR DBD lever arm region and show how DNA allosterically controls GR structure and dynamics.

Sequences flanking the core-binding site modulate glucocorticoid receptor structure and activity.

The glucocorticoid receptor (GR) binds as a homodimer to genomic response elements, which have particular sequence and shape characteristics. Here we show that the nucleotides directly flanking the core-binding site, differ depending on the strength of GR-dependent activation of nearby genes. Our study indicates that these flanking nucleotides change the three-dimensional structure of the DNA-binding site, the DNA-binding domain of GR and the quaternary structure of the dimeric complex. Functional studies in a defined genomic context show that sequence-induced changes in GR activity cannot be explained by differences in GR occupancy. Rather, mutating the dimerization interface mitigates DNA-induced changes in both activity and structure, arguing for a role of DNA-induced structural changes in modulating GR activity. Together, our study shows that DNA sequence identity of genomic binding sites modulates GR activity downstream of binding, which may play a role in achieving regulatory specificity towards individual target genes.

Natural disordered sequences in the amino terminal domain of nuclear receptors: lessons from the androgen and glucocorticoid receptors.

Steroid hormones are a diverse class of structurally related molecules, derived from cholesterol, that include androgens, estrogens, progesterone and corticosteroids. They represent an important group of physiologically active signalling molecules that bind intracellular receptor proteins and regulate genes involved in developmental, reproductive and metabolic processes. The receptor proteins share structurally and functionally related ligand binding and DNA-binding domains, but possess distinct N-terminal domains (NTD) of unique length and amino acids sequence. The NTD contains sequences important for gene regulation, exhibit structure plasticity and are likely to contribute to the specificity of the steroid hormone/receptor response.

Nitric oxide up-regulates the glucocorticoid receptor and blunts the inflammatory reaction in porcine endotoxin sepsis.

OBJECTIVES: Nitric oxide inhibits the expression of many genes involved in inflammatory diseases. Glucocorticoids inhibit similar transcription factors. We hypothesized that there may be an interaction between nitric oxide and glucocorticoids, with the potential to enhance the anti-inflammatory effect when administered simultaneously. DESIGN: Prospective, randomized, controlled study. SETTING: Animal research laboratory. SUBJECTS: A total of 45 anesthetized and mechanically ventilated pigs. INTERVENTIONS: Lung and systemic injury was induced by intravenous infusion of endotoxin (lipopolysaccharide) for 6 hrs. After 2.5 hrs, one group received 3.5 mg/kg hydrocortisone, another group inhaled nitric oxide (30 ppm), and still another group received both steroid and nitric oxide. Control groups of healthy and endotoxin-exposed piglets were also studied. MEASUREMENTS AND MAIN RESULTS: Central hemodynamics and gas exchange were measured. Detection of the glucocorticoid receptor and inflammatory markers in lung, liver, and kidney tissue were made by immunohistochemistry, and morphology was studied with light microscopy. Endotoxin infusion markedly reduced glucocorticoid receptor expression in lung, liver, and kidney and up-regulated activator protein-1 and the inflammatory markers nuclear factor-kappaB and tumor necrosis factor-alpha. When administered separately, steroids and nitric oxide had modest effect on the inflammatory response. However, nitric oxide up-regulated the glucocorticoid receptor expression. Simultaneous administration of steroids and nitric oxide attenuated the inflammatory response and almost preserved or restored normal histology of both lung and systemic organs. When the glucocorticoid receptor was blocked by a receptor antagonist (mifepristone, 600 mg) and inhaled nitric oxide was subsequently administered, no increase in the expression of the glucocorticoid receptor was seen. CONCLUSION: We suggest that up-regulation of glucocorticoid receptor expression by nitric oxide made steroid therapy more effective.

Analysis of cellular functions by multipoint fluorescence correlation spectroscopy.

The biophysical investigation of living cells is currently possible by single molecular detection methods such as fluorescence correlation spectroscopy (FCS). FCS is applied for measuring the dynamic mobility of target molecules in living cells; however, the conventional FCS systems still lack quantitative analysis for many regions of interests (ROI) in real time. To improve this situation, we have developed a novel multipoint FCS system (M-FCS) that can measure multipoint correlation functions in the cell simultaneously. To evaluate its performance, we measured correlation functions for rhodamine 6G (Rh6G) in homogeneous conditions and for green fluorescence protein (GFP) in HeLa cells. We conclude that M-FCS possesses reliable performance. As a pharmacological application, glucocorticoid receptor protein fused GFP (GR-GFP) was transfected in HeLa cells and FCS measurements were carried out in the cytoplasm and the nucleus simultaneously. The translocation of GR-GFP from the cytoplasm to the nucleus by ligand stimulation was observed with laser scanning microscopy (LSM) and M-FCS. Particularly in the nucleus, the slower diffusion of GR-GFP suggested molecular interactions after the translocation. These data imply that M-FCS can be applied for quantitative analysis of kinetic processes in living cells.

The dynamic localization of the glucocorticoid receptor in rat C6 glioma cell mitochondria.

Glucocorticoids modify gene expression via the translocation of receptors from the cytosol to the nucleus following agonist-associated receptor activation. In this study, we have characterized mitochondrial glucocorticoid (GR) localization and associated translocation kinetics in the C6 mouse glioma cell line. Treatment of the cells, which were cultured in steroid-depleted culture medium, with the GR agonist dexamethasone (dex) resulted in a dramatic decrease in mitochondrial GR levels in parallel with those of the cytosolic receptor. The effect was not observed in isolated intact mitochondria suggesting that the effect is unlikely to be direct but is rather a component of the combined cellular response to GR activation. A marked stimulation of the expression of the mitochondrially-encoded cytochrome oxidase-1 (COX-1) gene was found following GR activation and its export from mitochondria. The effects were inhibited by RU486. Therefore, GR is likely to have a functional role at the level of the mitochondria within intact cells.

Visualization and mechanism of assembly of a glucocorticoid receptor.Hsp70 complex that is primed for subsequent Hsp90-dependent opening of the steroid binding cleft.

A minimal system of five proteins, hsp90, hsp70, Hop, hsp40, and p23, assembles glucocorticoid receptor (GR).hsp90 heterocomplexes and causes the simultaneous opening of the steroid binding cleft to access by steroid. The first step in assembly is the ATP-dependent and hsp40 (YDJ-1)-dependent formation of a GR.hsp70 complex that primes the receptor for subsequent ATP-dependent activation by hsp90, Hop, and p23. This study focuses on three aspects of the GR priming reaction with hsp70. First, we have visualized the primed GR.hsp70 complexes by atomic force microscopy, and we find the most common stoichiometry to be 1:1, with some complexes of a size approximately 1:2 and a few complexes of larger size. Second, in a recent study of progesterone receptor priming, it was shown that hsp40 binds first, leading to the notion that it targets hsp70 to the receptor. We show here that hsp40 does not perform such a targeting function in priming the GR. Third, we focus on a short amino-terminal segment of the ligand binding domain that is required for GR.hsp90 heterocomplex assembly. By using two glutathione S-transferase (GST)/ligand binding domain fusions with (GST/520C) and without (GST/554C) hsp90 binding and steroid binding activity, we show that the priming step with hsp70 occurs with GST/554C, and it is the subsequent assembly step with hsp90 that is defective.

Localization of glucocorticoid receptors in the murine inner ear.

We performed an immunohistochemical investigation of the distribution of glucocorticoid receptors (GRs) in the murine inner ear and found that GRs were expressed extensively, but with various degrees of immunoreactivity in different regions. We observed the strongest GR expression in the type III fibrocytes of the spiral ligament. Although the immunoreactivity of the cochlear hair cells and of the vestibular sensory epithelia was weak, the neighboring cochlear supporting cells and the subepithelial regions of the vestibular sensory epithelia were immunostained. Staining for GRs was also positive in the spiral ganglia and vestibular ganglia, as well as in the endolymphatic sac. The role of GRs in the inner ear is discussed.

Subcellular distribution of the glucocorticoid receptor and evidence for its association with microtubules.

The cellular distribution of the glucocorticoid receptor (GR) has not yet been firmly established. The extensive literature indicates that GR is present both in the cytoplasm and the cell nucleus, however, some studies have failed to detect cytoplasmic GR. It is still controversial as to whether GR is randomly diffusing in the cytoplasm and nucleus, or if the GR-distribution is organized or controlled in some way, which may be of importance for the transduction of glucocorticoid effects to cells. There is evidence that both non-activated and activated GR is associated with the plasma membrane, a number of cytoplasmic organelles and the nucleus. Both morphological and biochemical evidence show that GR is associated with microtubules during different stages of the cell cycle, i.e. GR co-localizes, co-purifies and co-polymerizes with tubulin. This indicates that GR is structurally linked to the intracellular MT-network which may be of importance in the mechanism of action of glucocorticoid hormones. The literature in this field is reviewed including the reported data on subcellular GR-localization.

A comparison of 15N NMR relaxation measurements with a molecular dynamics simulation: backbone dynamics of the glucocorticoid receptor DNA-binding domain.

The rapid motions of the backbone of the DNA-binding domain of the glucocorticoid receptor (GR DBD) have been investigated using proton-detected heteronuclear NMR experiments on 15N-labeled protein at pH 6.0 and with a 200 psec molecular dynamics simulation of hydrated GR DBD. The experimental data were interpreted in terms of a generalized order parameter (S2) and an effective correlation time (tau e) for the internal motion of each amide bond. A back calculation, using the same model, yielded the [1H]-14N nuclear Overhauser effects (NOEs) and the 15N spin-lattice relaxation times (T1) from the simulated data. The rapid motions of the backbone turned out to be rather limited and uniform throughout the protein, with a somewhat reduced mobility in the two major alpha-helical regions and a slightly enhanced flexibility for some residues in the first zinc coordinating region. The agreement between the experimental and simulated S2-values was as good as quantitative for most of the residues, except for some residues that were subject to a more large-scale, and in the simulation thus poorly sampled, motion. Examples of such motions that were found in the simulation include jumps of the amide bond of Ile-487 between the charged oxygens of the side chain of Asp-485 and less distinct large scale motions for some of the residues in the extended regions, that were shown to give rise to noisy and/or fast decaying internal reorientational correlation functions. For these residues large differences in the simulated and experimental tau e-values were found, indicating that motions on different time scales were dominating in the experimental and simulated values. The lower (< 0.7) experimental NOEs for these residues could not be reproduced in the simulation and were shown to be a consequence of the lower tau e-values estimated in the simulation. By combining information from the simulation and the experiment a more complete picture of the motions for these residues can be obtained as is illustrated with an estimation of the jump angle and jump frequency for the amide bond of Ile-487.

Electron microscopic demonstration of glucocorticoid recognition sites on isolated rat hepatocytes.

Ultrastructural evidence is presented for the presence of membrane-bound glucocorticoid recognition and binding sites. Corticosterone was derivatized at 3 different positions and coupled covalently to bovine serum albumin (BSA). All three derivatives competed for binding of [3H]corticosterone by isolated rat hepatocytes. The most effective competitor, corticosterone-succinate-BSA (CSB), was adsorbed onto colloidal gold particles (CSB-gold, 17 +/- 3 nm dia). When isolated rat hepatocytes or mouse pituitary tumor cells (AtT 20) are incubated with CSB-gold, specific binding in the microvilli-rich region of these cells is seen. This binding of CSB-gold is reduced by about 50% in the presence of unlabelled CSB or corticosterone.

The glucocorticoid receptor in homodimeric and monomeric form visualised by electron microscopy.

The purified glucocorticoid receptor (GR) from rat liver has been visualised by electron microscopy. The specimens were prepared by spreading on thin carbon support and negatively stained using uranyl acetate. Two forms of GR, the monomeric and the dimeric forms, were identified based on size, chromatographic distribution, and DNA binding properties. The GR monomer consists of two globular domains of slightly different size with a thinner connecting domain in between. In the absence of DNA the dimeric GR has a characteristic four-leaf clover structure. The size and appearance of this structure is consistent with two GR subunits arranged in a side-by-side fashion. Monomeric and dimeric GR specifically bound to DNA are also shown.

Structural differences between the glucocorticoid, dioxin and oxysterol receptors from rat liver cytosol.

The rat hepatic glucocorticoid, dioxin and oxysterol receptors were subjected to high performance liquid chromatography on size-exclusion and anion-exchange columns. Both the glucocorticoid receptor and the dioxin receptor had a Stokes radius Rs approximately 7.5 nm, expected value for heteromeric complexes containing a dimer of the Mr approximately 90,000 heat shock protein, hsp90 (Rs approximately 7.0 nm). The oxysterol receptor represented a much smaller entity (Rs approximately 6.0 nm). When analyzed on a Mono Q anion-exchange column, the molybdate-stabilized glucocorticoid receptor and dioxin receptor eluted as single peaks at approximately 0.30 M and 0.26-0.28 M NaCl, respectively, whereas the oxysterol receptor represented a less negatively charged species (0.11-0.14 M NaCl). Following washing of the Mono Q column with molybdate-free buffer, the activated monomeric glucocorticoid receptor was detected (0.10-0.12 M NaCl). In contrast, no modification in the elution pattern of the dioxin receptor and the oxysterol receptor was observed. These data demonstrate differences in the physico-chemical properties of the glucocorticoid, dioxin and oxysterol receptors, respectively, which might reflect structural differences.

Unspecific DNA binding of the DNA binding domain of the glucocorticoid receptor studied with flow linear dichroism.

The unspecific interaction between the DNA-binding domain of the human glucocorticoid receptor and DNA was studied using linear dichroism (LD) and circular dichroism (CD) spectroscopy. The amplitude of the LD signal was found to increase upon addition of protein at ionic strengths less than 60 nM Na+, indicating an increased persistence length of the complex compared to uncomplexed DNA. Analysis of the LD spectrum suggests that the binding does not involve intercalation of tyrosine residues. Evidence of saturation is found at a binding stoichiometry of approximately 5 DNA base pairs per protein monomer.

Two amino acids within the knuckle of the first zinc finger specify DNA response element activation by the glucocorticoid receptor.

The specificity of target gene activation by steroid receptors is encoded within a small, cysteine-rich domain that is believed to form two zinc-coordinated fingers. Here we show that the ability of glucocorticoid and estrogen receptors to discriminate between their closely related response elements resides in the two amino acids located between the two cysteines in the C-terminal half of the first finger. Unexpectedly, chimeric glucocorticoid receptors harboring portions of the interfinger and/or second finger of the estrogen receptor have the ability to activate transcription from either a GRE- or ERE-containing promoter. We surmise that whereas the "knuckle" region of the first finger may be the primary determinant of sequence recognition, the remainder of the DNA binding domain normally confers structural information required for preventing promiscuous HRE recognition.

Direct evidence for intra- and intermolecular disulfide bond formation in the human glucocorticoid receptor. Inhibition of DNA binding and identification of a new receptor-associated protein.

We have investigated the potential for the steroid affinity-labeled human glucocorticoid receptor to form both intramolecular and intermolecular disulfide bonds. Glucocorticoid receptors labeled in intact HeLa S3 cells with the covalent affinity label [3H]dexamethasone mesylate ([3H]DM) were analyzed on denaturing 5-12% polyacrylamide gels under both nonreducing and reducing conditions. Under nonreducing conditions the affinity-labeled receptor migrated as a heterogeneous species having an average molecular mass of approximately 96 kDa whereas, under reducing conditions, the receptor migrated as a more discrete form. These data suggest that a reducing environment can influence the structure of the glucocorticoid receptor monomer and further imply that sulfhydryl groups within the affinity-labeled receptor are available for modification. To pursue this observation in greater detail, we tested the effect of oxidizing conditions on the structure of the glucocorticoid receptor. The presence of low concentrations (0.125-0.5 mM) of three oxidizing reagents (sodium tetrathionate, disulfiram, and iodosobenzoate) altered the migration of the affinity-labeled receptor resulting in forms of apparent lower molecular mass (as low as 78 kDa). This altered migration, not seen with most other cytosolic proteins, is consistent with the formation of intramolecular disulfide bonds within the receptor which presumably cause it to assume a folded conformation and migrate faster through the gel. At higher concentrations of these reagents (up to 5.0 mM), we also detect a saturably labeled [3H]DM band which has a higher molecular mass (approximately 140 kDa), indicating the formation of intermolecular disulfide bonds between the [3H]DM-labeled receptor and another closely associated protein(s) having a molecular mass of approximately 40 kDa. The effects which these oxidizing reagents have on glucocorticoid receptor structure are completely reversed upon the addition of dithiothreitol, indicating that the observed changes in migration do not reflect receptor proteolysis but rather a folding and unfolding within the receptor monomeric protein. We have also analyzed the effect of this oxidation/reduction on the function of the glucocorticoid receptor. Oxidation of the [3H]DM-labeled receptor complex with 0.5 mM sodium tetrathionate inhibited activation of receptor to a form capable of binding to DNA-cellulose. This inhibition can be reversed with dithiothreitol at 25 degrees C but not at 0 degrees C, suggesting that these oxidizing reagents are inhibitory at the transformation and/or activation steps.(ABSTRACT TRUNCATED AT 400 WORDS)