A conformational switch in the ligand-binding domain regulates the dependence of the glucocorticoid receptor on Hsp90.

Steroid hormone receptors (SRs) are transcription factors that act as regulatory switches by altering gene expression in response to ligands. The highly conserved ligand-binding domain of SRs is a precise but versatile molecular switch that can adopt distinct conformations. Differential stabilization of these conformations by ligands, DNA response elements and transcriptional coregulators controls the activity of SRs in a gene-specific and cell-specific manner. In the case of the glucocorticoid receptor (GR), high-affinity ligand binding requires the interaction of the LBD with the heat shock protein 90 (Hsp90). Here, we show that the dependence of the ligand binding ability of GR on Hsp90 can be modified by the replacement of single amino acids within an allosteric network that connects the buried ligand-binding pocket and a solvent-exposed coregulator interaction surface. Each of the identified mutations altered the equilibrium between alternative GR conformations distinctively, indicating that the Hsp90 dependence of SRs may correlate with differences in the conformational dynamics of these receptors. Our results suggest that Hsp90 stabilizes the GR ligand-binding pocket indirectly by utilizing the allosteric network, while allowing the receptor to remain structurally uncommitted. Thus, in addition to ensuring the accessibility of the GR ligand-binding pocket to ligands, Hsp90 seems to enable hormones and coregulators to act as allosteric effectors, which forms the basis for gene-specific and cell-specific responses of GR to ligands.

The catalytic mechanism of indole-3-glycerol phosphate synthase: crystal structures of complexes of the enzyme from Sulfolobus solfataricus with substrate analogue, substrate, and product.

Indoleglycerol phosphate synthase catalyzes the ring closure of an N-alkylated anthranilate to a 3-alkyl indole derivative, a reaction requiring Lewis acid catalysis in vitro. Here, we investigated the enzymatic reaction mechanism through X-ray crystallography of complexes of the hyperthermostable enzyme from Sulfolobus solfataricus with the substrate 1-(o-carboxyphenylamino) 1-deoxyribulose 5-phosphate, a substrate analogue and the product indole-3-glycerol phosphate. The substrate and the substrate analogue are bound to the active site in a similar, extended conformation between the previously identified phosphate binding site and a hydrophobic pocket for the anthranilate moiety. This binding mode is unproductive, because the carbon atoms that are to be joined are too far apart. The indole ring of the bound product resides in a second hydrophobic pocket adjacent to that of the anthranilate moiety of the substrate. Although the hydrophobic moiety of the substrate moves during catalysis from one hydrophobic pocket to the other, the triosephosphate moiety remains rigidly bound to the same set of hydrogen-bonding residues. Simultaneously, the catalytically important residues Lys53, Lys110 and Glu159 maintain favourable distances to the atoms of the ligand undergoing covalent changes. On the basis of these data, the structures of two putative catalytic intermediates were modelled into the active site. This new structural information and the modelling studies provide further insight into the mechanism of enzyme-catalyzed indole synthesis. The charged epsilon-amino group of Lys110 is the general acid, and the carboxylate group of Glu159 is the general base. Lys53 guides the substrate undergoing conformational transitions during catalysis, by forming a salt-bridge to the carboxylate group of its anthranilate moiety.

Novel glucocorticoid receptor coactivator effector mechanisms.

Glucocorticoids regulate numerous distinct physiological processes, most of which rely on the ability of the hormone-bound glucocorticoid receptor (GR) to change the expression of target genes in a cell- and promoter-dependent manner. The transcriptional activity of GR depends on coactivators that regulate transcription by remodeling chromatin or by facilitating the recruitment of the basal transcriptional machinery. Coactivators are often part of multiprotein complexes that are not specific for GR but also mediate the activity of other nuclear receptors (NRs) and unrelated transcription factors. Surprisingly, recent results reveal that the activity of coactivators might contribute to the receptor, promoter and cell specificity of NR action. The emerging picture shows coactivators as flexible, but precise, coordinators of complex and dynamic networks, in which transcriptional regulation by GR and other NRs is linked to other signaling pathways.

Phosphorylation of the nuclear receptor SF-1 modulates cofactor recruitment: integration of hormone signaling in reproduction and stress.

Steroidogenic factor 1 (SF-1) is an orphan nuclear receptor that serves as an essential regulator of many hormone-induced genes in the vertebrate endocrine system. The apparent absence of a SF-1 ligand prompted speculation that this receptor is regulated by alternative mechanisms involving signal transduction pathways. Here we show that maximal SF-1-mediated transcription and interaction with general nuclear receptor cofactors depends on phosphorylation of a single serine residue (Ser-203) located in a major activation domain (AF-1) of the protein. Moreover, phosphorylation-dependent SF-1 activation is likely mediated by the mitogen-activated protein kinase (MAPK) signaling pathway. We propose that this single modification of SF-1 and the subsequent recruitment of nuclear receptor cofactors couple extracellular signals to steroid and peptide hormone synthesis, thereby maintaining dynamic homeostatic responses in stress and reproduction.

An additional region of coactivator GRIP1 required for interaction with the hormone-binding domains of a subset of nuclear receptors.

Transcriptional coactivators of the p160 family (SRC-1, GRIP1, and p/CIP) associate with DNA-bound nuclear receptors (NRs) and help the NRs to recruit an active transcription initiation complex to the promoters of target genes. Previous studies have demonstrated the importance of the NR interaction domain (NID) of p160 proteins containing three NR box motifs (LXXLL) for the interaction with the hormone-binding domains of NRs. Here we report that, in addition to NID, another region of coactivator GRIP1 (amino acids 1011-1121), called the auxiliary NID (NIDaux), is required in vitro and in vivo for efficient interaction with a subset of NRs, including the glucocorticoid receptor (GR), androgen receptor, and retinoic acid receptor alpha. A second group of NRs, which includes the progesterone receptor, retinoid X receptor alpha, thyroid hormone receptor beta1, and vitamin D receptor, required only NID for efficient interaction. For binding to GR, the NID and NIDaux of GRIP1 must act in cis, but deletion of up to 144 amino acids between the two regions did not reduce binding efficiency. Amino acids 1011-1121 of GRIP1 also contain a p300 interaction domain, but mutational analysis indicated that the p300 interaction function within this region is separable from the ability to contribute to GR hormone-binding domain binding. SRC-1 lacks an NIDaux activity equivalent to that in GRIP1.

Structure and specificity of nuclear receptor-coactivator interactions.

Combinatorial regulation of transcription implies flexible yet precise assembly of multiprotein regulatory complexes in response to signals. Biochemical and crystallographic analyses revealed that hormone binding leads to the formation of a hydrophobic groove within the ligand binding domain (LBD) of the thyroid hormone receptor that interacts with an LxxLL motif-containing alpha-helix from GRIP1, a coactivator. Residues immediately adjacent to the motif modulate the affinity of the interaction; the motif and the adjacent sequences are employed to different extents in binding to different receptors. Such interactions of amphipathic alpha-helices with hydrophobic grooves define protein interfaces in other regulatory complexes as well. We suggest that these common structural elements impart flexibility to combinatorial regulation, whereas side chains at the interface impart specificity.