Structure refinement of the glucocorticoid receptor-DNA binding domain from NMR data by relaxation matrix calculations.

The solution structure of the glucocorticoid receptor (GR) DNA-binding domain (DBD), consisting of 93 residues, has been refined from two and three-dimensional NMR data using an ensemble iterative relaxation matrix approach followed by direct NOE refinement with DINOSAUR. A set of 47 structures of the rat GR fragment Cys440-Arg510 was generated with distance geometry and further refined with a combination of restrained energy minimization and restrained molecular dynamics in a parallel refinement protocol. Distance constraints were obtained from an extensive set of NOE build-up curves in H2O and 2H2O via relaxation matrix calculations (1186 distance constraints from NOE intensities, 10 phi and 22 chi 1 dihedral angle constraints from J- coupling data were used for the calculations). The root-mean-square deviation values of the 11 best structures on the well-determined part of the protein (Cys440 to Ser448, His451 to Glu469 and Pro493 to Glu508) are 0.60 A and 1.20 A from the average for backbone and all heavy atoms, respectively. The final structures have R-factors around 0.40 and good stereochemical qualities. The first zinc-coordinating domain of the GR DBD is very similar to the crystal structure with a root-mean-square difference of 1.4 A. The second zinc-coordinating domain is still disordered in solution. No secondary structure element is found in this domain in the free state. As suggested by crystallographic studies on the estrogen receptor DBD-DNA and GR DBD-DNA complexes, part of this region will form a distorted helix and the D-box will undergo a conformational change upon cooperative binding to DNA.

1H NMR studies of DNA recognition by the glucocorticoid receptor: complex of the DNA binding domain with a half-site response element.

The complex of the rat glucocorticoid receptor (GR) DNA binding domain (DBD) and half-site sequence of the consensus glucocorticoid response element (GRE) has been studied by two-dimensional 1H NMR spectroscopy. The DNA fragment is a 10 base-pair oligonucleotide, 5'd(GCTGTTCTGC)3'.5'd-(GCAGAACAGC)3', containing the stronger binding GRE half-site hexamer, with GC base pairs at each end. The 93-residue GR-DBD contains an 86-residue segment corresponding to residues 440-525 of the rat GR. Eleven NOE cross peaks between the protein and DNA have been identified, and changes in the chemical shift of the DNA protons upon complex formation have been analyzed. Using these protein-DNA contact points, it can be concluded that (i) the "recognition helix" formed by residues C460-E469 lies in the major groove of the DNA; (ii) the GR-DBD is oriented on the GRE half-site such that residues A477-D481, forming the so-called D-loop, are available for protein-protein interaction in the GR-DBD dimer on the intact consensus GRE; and (iii) the 5-methyl of the second thymine in the half-site and valine 462 interact, confirming indirect evidence [Truss et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7180-7184; Mader et al. (1989) Nature 338, 271-274] that both play an important role in GR-DBD DNA binding. These findings are consistent with the model proposed by Härd et al. [(1990) Science 249, 157-160] and the X-ray crystallographic complex structure determined by Luisi et al. [(1991) Nature 352, 497-505].

Identification of the metal coordinating residues in the DNA binding domain of the glucocorticoid receptor by 113Cd-1H heteronuclear NMR spectroscopy.

Two-dimensional 1H-113Cd HSQC and relay HSQC experiments were performed on the 113Cd substituted DNA binding domain of the rat glucocorticoid receptor. The results of these experiments combined with sequence-specific assignments allowed the identification of all coordinating cysteines. It was found that C495 and not C500 is the fourth coordinating cysteine in the second zinc-finger. A signal at approximately 2 ppm previously assigned to a epsilon-CH3 of a methionine residue coordinating to a third, weakly bound, cadmium ion, was identified as the C443 beta proton ligating to the metal ion in the first zinc-finger. No indications were found for the presence of a previously suggested third metal ion binding site.

Photo-CIDNP study of the interaction between the glucocorticoid receptor DNA-binding domain and glucocorticoid response elements.

Photo-CIDNP studies were performed on two protein fragments that both contain the double zinc-finger DNA-binding domain of the glucocorticoid receptor. In the absence of DNA, Tyr452 and Tyr474 are polarised in both fragments while Tyr497 is not. Addition of a palindromic glucocorticoid response element (GRE) results in the suppression of Tyr474 polarization while the polarization of Tyr452 is unaffected. The same result is observed upon adding a half GRE to the protein fragment indicating that the suppression of Tyr474 polarization is not due to protein-protein contacts but to interaction with DNA.

1H NMR studies of the glucocorticoid receptor DNA-binding domain: sequential assignments and identification of secondary structure elements.

Two protein fragments containing the DNA-binding domain (DBD) of the glucocorticoid receptor (GR) have been studied by two-dimensional 1H NMR spectroscopy. The two peptides (93 and 115 residues, respectively) contain a common segment corresponding to residues C440-I519 of the rat GR or residues C421-I500 of the human GR and include two Zn-binding "finger" domains. The structures of this segment are almost identical in the two protein fragments, as judged from chemical shifts and sequential NOE connectivities. More than 90% of all observable 1H resonances within a 71-residue segment encompassing C440-R510 (rat GR) could be sequentially assigned by standard techniques, and stereospecific assignments could be made for the methyl groups in four valine residues within this segment. Sequential NOE connectivities indicate several elements of secondary structure including two alpha-helical segments consisting of residues S459-E469 and P493-G504, a type I reverse turn between residues R479 and C482, a type II reverse turn between residues L475 and G478, and several regions of extended peptide conformation. No evidence for alpha-helical conformation was found within the two putative zinc-finger domains, indicating that the structures of these domains differ from that of TFIIIA-type zinc fingers. The observation of some very slowly exchanging amide protons in the N-terminal (CI) domain of the DBD in combination with slow rotation of the Y452 aromatic ring indicates that this domain has a restricted conformational flexibility compared to the C-terminal (CII) domain. We also observe several long-range NOE connectivities within C440-R510, suggesting that the sequential assignments presented here will provide a basis for a complete structure determination of this segment of the GR.

Solution structure of the glucocorticoid receptor DNA-binding domain.

The three-dimensional structure of the DNA-binding domain (DBD) of the glucocorticoid receptor has been determined by nuclear magnetic resonance spectroscopy and distance geometry. The structure of a 71-residue protein fragment containing two "zinc finger" domains is based on a large set of proton-proton distances derived from nuclear Overhauser enhancement spectra, hydrogen bonds in previously identified secondary structure elements, and coordination of two zinc atoms by conserved cysteine residues. The DBD is found to consist of a globular body from which the finger regions extend. A model of the dimeric complex between the DBD and the glucocorticoid response element is proposed. The model is consistent with previous results indicating that specific amino acid residues of the DBD are involved in protein-DNA and protein-protein interactions.

Glucocorticoid receptor mutants that define a small region sufficient for enhancer activation.

Transcriptional enhancement is a general mechanism for regulation of gene expression in which particular proteins bound to specific DNA sequences stimulate the efficiency of initiation from linked promoters. One such protein, the glucocorticoid receptor, mediates enhancement in a glucocorticoid hormone-dependent manner. In this study, a region of the 795-amino acid rat glucocorticoid receptor that is active in transcriptional enhancement was identified. The active region was defined by expressing various receptor deletion mutants in stably and transiently transfected cells and examining the regulated transcription of hormone-responsive genes. Mutant receptors lacking as many as 439 amino-terminal amino acids retained activity, as did those with as many as 270 carboxyl-terminal amino acids deleted. This suggests that the 86-amino acid segment between the most extensive terminal deletions, which also includes sequences required for specific DNA binding in vitro, is sufficient for enhancer activation. In fact, a 150-amino acid receptor fragment that encompasses this segment mediates constitutive enhancement.

Intracellular receptor concentration limits glucocorticoid-dependent enhancer activity.

The glucocorticoid receptor protein, in association with cognate hormonal ligands, binds with high affinity to specific DNA sequences termed glucocorticoid response elements (GREs) which can function as hormone-dependent transcriptional enhancers; thus, the receptor is a regulable enhancer-activating protein. We have constructed cell lines expressing different levels of glucocorticoid receptor, and demonstrate that the extent of a structural alteration in the chromatin at a characterized GRE, as well as the magnitude of several transcriptional responses elicited by the receptor, are roughly proportional to the number of receptor molecules per cell. Thus, for three independent glucocorticoid-responsive transcription units examined in our HTC-derived cell lines, the receptor appears to be a primary regulatory factor. Moreover, the results suggest that other cellular factors required for the assembly and function of GREs and transcription initiation complexes must be produced in excess relative to their levels of utilization at normal receptor concentrations.

Genetic complementation of a glucocorticoid receptor deficiency by expression of cloned receptor cDNA.

We isolated and sequenced 6.3 kb of cDNA encoding that rat glucocorticoid receptor, a protein that binds and activates a class of hormone-dependent transcriptional enhancers. Receptor-containing cells produce receptor mRNAs of approximately equal to 6.5 kb and approximately equal to 4.8 kb that differ only in their 3' nontranslated regions; an open reading frame of 795 amino acids resides within the 5' portion of the transcripts. The coding region was expressed in vitro, in transient transfections, and in stable transfectants of a receptor-deficient cell line. The protein products are indistinguishable from bona fide receptor with respect to sedimentation and electrophoretic mobility, antibody reactivity, and hormone and DNA binding. Moreover, the cloned receptor protein activates its corresponding enhancers, restoring to the receptor-deficient cells the full capacity for regulated enhancement.

DNA sequences bound specifically by glucocorticoid receptor in vitro render a heterologous promoter hormone responsive in vivo.

Glucocorticoids stimulate transcriptional initiation within integrated mammary tumor virus (MTV) DNA sequences in infected cells. We report here that production of herpes simplex virus thymidine kinase (tk) RNA is stimulated as much as 50-fold by the glucocorticoid, dexamethasone, when sequences from a particular region of MTV DNA are fused upstream of the normally constitutive tk promoter region. Three cloned fragments of MTV DNA were tested, each itself lacking the sequences required for transcription initiation. We monitored the effects of dexamethasone on the efficiency with which these recombinants transfect a tk- rat cell line to a tk+ phenotype, and measured tk enzymatic activity, the size and abundance of tk mRNA, and the 5' termini of tk transcripts in the transfectants. We conclude that the MTV promoter region contains a "glucocorticoid response element" that can be separated from a second element essential for MTV transcription initiation. The hormone response element maps within a 340-base pair MTV DNA fragment that contains specific binding sites for purified glucocorticoid receptor protein in vitro, implying that receptor binding at these sites in vivo may mediate hormone responsiveness. Comparison of several different constructions indicates that the location and orientation of the glucocorticoid response element relative to the transcription start site is not rigidly constrained.

Phosphoribosylpyrophosphate synthetase is elevated in fibroblasts from patients with the Lesch-Nyhan syndrome.

In subconfluent cultures of fibroblasts from patients with complete or partial deficiencies of hypoxanthine-guanine phosphoribosyltransferase, phosphoribosylpyrophosphate synthetase activity is elevated. The abnormally high catalytic activity of the synthetase appears to account for the overproduction of purines by the cultured mutant cells and presumably for that by the patients.