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ABSTRACT: Estrogens exert their effects on target tissues by binding to a nuclear transcription factor termed the estrogen receptor (ER). Previous structural studies have demonstrated that each class of ER ligand (agonist, partial agonist, and SERM antagonist) induces distinctive orientations in the receptor's carboxy-terminal transactivation helix. The conformation of this portion of the receptor determines whether ER can recruit and interact with the components of the transcriptional machinery, thereby facilitating target gene expression.
We have determined the structure of rat ERbeta ligand binding domain (LBD) in complex with the pure antiestrogen ICI 164,384 at 2.3 A resolution. The binding of this compound to the receptor completely abolishes the association between the transactivation helix (H12) and the rest of the LBD. The structure reveals that the terminal portion of ICI's bulky side chain substituent protrudes from the hormone binding pocket, binds along the coactivator recruitment site, and physically prevents H12 from adopting either its characteristic agonist or AF2 antagonist orientation.
The binding mode adopted by the pure antiestrogen is similar to that seen for other ER antagonists. However, the size and resultant positioning of the ligand's side chain substituent produces a receptor conformation that is distinct from that adopted in the presence of other classes of ER ligands. The novel observation that binding of ICI results in the complete destabilization of H12 provides some indications as to a possible mechanism for pure receptor antagonism.
Structure 03/2001; 9(2):145-53. · 6.35 Impact Factor
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ABSTRACT: Here we summarise the results that have emerged from our structural studies on the oestrogen receptor (ER) ligand-binding domain. We have investigated the conformational effects of a variety of ligands on the structures of both ER isoforms. Each class of ligand (agonists, partial agonists and selective oestrogen receptor modulators) induces a unique conformation in the receptor's ligand-dependent transcriptional activation function. Together these studies have broadened our understanding of ER function by providing a unique insight into ER's ligand specificity and the structural changes that underlie receptor agonism and antagonism.
European Journal of Cancer 10/2000; 36 Suppl 4:S17-8. · 5.54 Impact Factor
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ABSTRACT: We have determined the three-dimensional structures of both alpha- and beta-forms of the ligand-binding domain of the oestrogen receptor (ER) in complexes with a range of receptor agonists and antagonists. Here, we summarize how these structures provide both an understanding of the ER's distinctive pharmacophore and a rationale for its ability to bind a diverse range of chemically distinct compounds. In addition, these studies provide a unique insight into the mechanisms that underlie receptor activation, as well as providing a structural basis for the antagonist action of molecules, such as raloxifene.
Biochemical Society Transactions 02/2000; 28(4):396-400. · 3.71 Impact Factor
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ABSTRACT: Oestrogens exert their physiological effects through two receptor subtypes. Here we report the three-dimensional structure of the oestrogen receptor beta isoform (ERbeta) ligand-binding domain (LBD) in the presence of the phyto-oestrogen genistein and the antagonist raloxifene. The overall structure of ERbeta-LBD is very similar to that previously reported for ERalpha. Each ligand interacts with a unique set of residues within the hormone-binding cavity and induces a distinct orientation in the AF-2 helix (H12). The bulky side chain of raloxifene protrudes from the cavity and physically prevents the alignment of H12 over the bound ligand. In contrast, genistein is completely buried within the hydrophobic core of the protein and binds in a manner similar to that observed for ER's endogenous hormone, 17beta-oestradiol. However, in the ERbeta-genistein complex, H12 does not adopt the distinctive 'agonist' position but, instead, lies in a similar orientation to that induced by ER antagonists. Such a sub-optimal alignment of the transactivation helix is consistent with genistein's partial agonist character in ERbeta and demonstrates how ER's transcriptional response to certain bound ligands is attenuated.
The EMBO Journal 10/1999; 18(17):4608-18. · 9.20 Impact Factor
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ABSTRACT: The overall architecture of the ligand binding domain (LBD) of members of the nuclear receptor superfamily are similar. There are now standard procedures to express and purify these proteins. A rapid and sensitive method for the structural analysis of these proteins is nano-electrospray tandem mass spectrometry. In the present study we have analysed the LBD of the human thyroid hormone receptor-beta-1 (TR-beta) by quadrupole time-of-flight tandem mass spectrometry. The intact protein was analysed in a carboxymethylated form in an attempt to identify which cysteine residues are located on the surface. The protein molecular weight (31 652.5 Da) was determined with an accuracy of +/-1 Da, while masses of tryptic fragments were determined with an accuracy of at least 75 ppm. The sequence coverage of the tryptic peptide mass map was 93.2 %. Tryptic peptides were subjected to collision-induced dissociation (CID) and the resulting product ions were mass measured with an accuracy of about 100 ppm. When accurate mass measurements were made with internal calibration, mass accuracies were improved to +/-2 ppm in mass spectra, and +/-20 ppm in CID spectra. From these data it was possible to determine the presence of post-translational modifications, locate the sites of carboxymethylation and, in addition, confirm the amino acid sequence of the expressed protein. To the best of our knowledge, this is the first characterisation of the TR-LBD-beta at the protein level.
Rapid Communications in Mass Spectrometry 02/1999; 13(18):1782-91. · 2.79 Impact Factor
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ABSTRACT: Oestrogens are involved in the growth, development and homeostasis of a number of tissues. The physiological effects of these steroids are mediated by a ligand-inducible nuclear transcription factor, the oestrogen receptor (ER). Hormone binding to the ligand-binding domain (LBD) of the ER initiates a series of molecular events culminating in the activation or repression of target genes. Transcriptional regulation arises from the direct interaction of the ER with components of the cellular transcription machinery. Here we report the crystal structures of the LBD of ER in complex with the endogenous oestrogen, 17beta-oestradiol, and the selective antagonist raloxifene, at resolutions of 3.1 and 2.6 A, respectively. The structures provide a molecular basis for the distinctive pharmacophore of the ER and its catholic binding properties. Agonist and antagonist bind at the same site within the core of the LBD but demonstrate different binding modes. In addition, each class of ligand induces a distinct conformation in the transactivation domain of the LBD, providing structural evidence of the mechanism of antagonism.
Nature 11/1997; 389(6652):753-8. · 36.28 Impact Factor
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ABSTRACT: Electrospray ionization mass spectrometry (ESMS) of the estrogen receptor ligand binding domain (ER LBD) in its estradiol-binding form was performed. A dimeric ER LBD was observed, with a greatly reduced capacity for protonation (major charge state for dimer +16 vs. +23 for a monomer). Peak broadening (probably due to heterogeneity resulting from salt and water adduct formation) adversely affected our ability to distinguish between multiple discreet dimeric species and thus prevented us from establishing an accurate average mass for the dimerized domain. A mixture of species with molecular masses between 57,240 Da and 57,900 Da was observed, which would compare to 57,274 Da, 57,546 Da, and 57,818 Da for the calculated masses of the dimer without estradiol, or with one or two bound ligand molecules, respectively. Hence, nonliganded ER LBD dimer appeared to constitute the major species. The presence of low levels of a singly liganded ER LBD dimer cannot be ruled out, but the data argue against the possibility of the ER LBD dimer carrying two molecules of estradiol. Allowing for current limitations in the technology, our data demonstrate that ESMS on a quadrupole mass spectrometer of limited mass range (4000 Da for singly charged ions) has potential utility for studying ligand-binding proteins. In particular, in future it might be possible to compare spectra obtained from agonist- and antagonist-bound receptors and determine from subtle changes in protonation state possible differences in the higher order structure of those noncovalent protein complexes.
Steroids 08/1996; 61(7):433-8. · 2.83 Impact Factor
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ABSTRACT: Experiments were carried out to determine the degree of solvent and reagent accessibility of the cysteines in the ligand-binding domain of the human estrogen receptor (hER LBD). The cysteine residues were alkylated when human ER LBD was present in its ligand (estradiol)-bound conformation. Direct electrospray ionization mass spectrometry (ESMS) as well as liquid chromatography coupled with ESMS, and matrix-assisted laser ionization desorption time-of-flight mass spectrometry were used to determine the location and the yield of the derivatized residues after proteolysis with trypsin. We observed that the cysteine 447 was protected against alkylation under these conditions, whereas cysteines 381, 417, and 530 were fully derivatized.
Steroids 07/1996; 61(6):367-73. · 2.83 Impact Factor
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ABSTRACT: A peptide hormone has been isolated from porcine intestine. Its primary structure was found to consist of 36 amino acid residues in a sequence identical to that of the porcine pancreatic polypeptide, previously not isolated from intestines or a tissue other than pancreas. The gut polypeptide significantly suppresses glucose-induced insulin secretion in vitro. Using an immunohistochemical technique, we also identified cells in the porcine gastrointestinal tract that were immunoreactive with pancreatic polypeptide. The immunoreactivity disappeared after absorption with the isolated gut polypeptide or synthetic human pancreatic polypeptide.
FEBS Letters 04/1994; 341(2-3):239-43. · 3.54 Impact Factor
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ABSTRACT: Two different forms of the monomeric NADP(+)-linked prostaglandin dehydrogenase/carbonyl reductase were purified from human placenta and shown to differ by the modification of a lysine residue. The modified and the unmodified proteins were reproducibly recovered in a ratio of approximately 1:3, and both were chemically stable. The modified form was more acidic (pI approximately 7.4 versus pI approximately 7.7) but indistinguishable from the unmodified form in specificity and activity. Amino acid analysis, sequence analysis, mass spectrometry, and chemical synthesis identified the modified residue as N6-(1-carboxyethyl)lysine with C-2 of propionic acid attached to the side-chain N of Lys-238. This compound can be formed from the lysine residue and pyruvate via a Schiff base and subsequent reduction. The enzyme and its NAD(+)-dependent counterpart are distantly related (23% residue identity) and have the same family assignment to short-chain dehydrogenases. Alignments and model-building into the tertiary structure of 3 alpha/20 beta-hydroxysteroid dehydrogenase show that carbonyl reductase has an extra loop (positions 149-189) that forms a separate extension and replaces a backbone C-terminal beta-strand. This change affects the substrate pocket, explaining the different substrate specificities but conserves residues of known functional importance. Carboxyethyllysine at position 238 corresponds to a proteolysis-sensitive position in several short-chain dehydrogenases, less well-defined in the model but close to a surface, and is compatible with the accessibility and enzyme properties observed.
Proceedings of the National Academy of Sciences 02/1993; 90(2):502-6. · 9.68 Impact Factor
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ABSTRACT: Cysteine is a relatively infrequent constituent of proteins, which in its thiol or half-cystine form contributes in a special manner to their three-dimensional structure. We show that in small cystine-containing peptides, the Cys content is always higher than the average in proteins in general. This observation makes it possible to search for new peptides by monitoring only their Cys content. We have developed a chemical assay for the detection of cyst(e)ine-rich peptides in tissue extracts. Using this assay we have isolated from porcine intestine a novel cysteine-rich peptide, which we denote ZF-1. ZF-1 is homologous to a single zinc-finger motif and has an acetylated N-terminus. This is the first demonstration of the existence of a processed single zinc-finger-like structure. The structural homology of ZF-1 to the zinc-finger motif, present in several metal-binding and DNA-binding proteins, suggests an important role of this peptide in metal transport and/or modulation of gene expression.
European Journal of Biochemistry 02/1993; 211(1-2):377-80. · 3.58 Impact Factor
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ABSTRACT: Liver alcohol dehydrogenase of the ethanol-active type ('class I enzyme') from the lizard, Uromastix hardwickii, was purified and screened for relationships with other vertebrate forms of the enzyme. Two different acetylated N-termini (acetyl-Gly and acetyl-Ser) and further positional differences already in the N-terminal segments establish the presence of two types of protein chain. The multiplicity is different from that hitherto detected within vertebrate class I alcohol dehydrogenase isozymes but typical of that which would be expected for subunits of different classes. In particular, relationships to class II or to class II-related forms appear likely. This may indicate yet further vertebrate alcohol dehydrogenase multiplicity or discovery of a class II non-mammalian enzyme. The results give prospects of defining gene duplications corresponding to more than one alcohol dehydrogenase class split to at an early vertebrate stage.
FEBS Letters 03/1992; 298(2-3):297-300. · 3.54 Impact Factor
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ABSTRACT: We recently isolated from pig intestine and characterized a 31-residue antibacterial peptide named cecropin-P1 with activity against Escherichia coli and several other Gram-negative bacteria. The isolation involved a number of batch-wise steps followed by several chromatography steps. The continued investigation of these antibacterial peptides has now yielded another antibacterial peptide with high activity against both E. coli and Bacillus megaterium. Amino acid analysis showed a very high content of proline (49 mol%) and arginine (26 mol%), an intermediate level of phenylalanine and low levels of leucine, tyrosine, isoleucine, and glycine. The primary structure was determined by a combination of Edman degradation, plasma desorption mass spectrometry and C-terminal sequence analysis by carboxypeptidase Y degradation using capillary zone electrophoresis for detection of liberated residues. The calculated molecular mass was 4719.7 Da, which is in excellent agreement with 4719 Da obtained by plasma desorption mass spectrometry. The peptide was named PR-39 (proline-arginine-rich with a size of 39 residues). The lethal concentration of the peptide was determined against six Gram-negative and four Gram-positive strains of bacteria.
European Journal of Biochemistry 01/1992; 202(3):849-54. · 3.58 Impact Factor
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ABSTRACT: Sorbin has been isolated from extracts of porcine upper intestine, and the biological activity in absorbing water and electrolytes utilized to monitor the purification procedure. Pure sorbin was obtained in a yield of about 1 mg/Mg boiled intestine. The protein chain has 153 amino acid residues and the primary structure was determined by analyses of CNBr-cleaved fragments and four enzymatic digests. The protein has a free N-terminal Met and an amidated C-terminal Ala. No structural similarity was observed with other known proteins in data bases, but several segments have special properties and the C-terminal half is rich in Pro and Arg.
European Journal of Biochemistry 11/1991; 201(1):53-9. · 3.58 Impact Factor
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ABSTRACT: We have isolated galanin from chicken intestine by monitoring for the N-terminal glycyltryptophan, which constitutes a conserved part characteristic of the peptide. This monitoring method complements that previously used for C-terminal amide detection and proves chemical monitoring of specific structures to be useful. The isolation allowed determination of the structure, which was found to be unidentical to any of the known galanins. However, N-terminal pentadecapeptide parts are identical, showing this segment to be of special importance. In addition to common substitutions at positions 16, 18, 23, 26 and 29, chicken galanin has phenylalanine at position 28, where all known mammalian galanins have leucine.
FEBS Letters 09/1991; 288(1-2):151-3. · 3.54 Impact Factor
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ABSTRACT: Dopamine was shown to affect Na+,K(+)-ATPase activity in basolateral membranes of the rat kidney via a pertussis toxin dependent mechanism. In order to examine if some form of pertussis toxin sensitive G-protein is present exclusively in the basolateral membrane of the rat renal cortex we examined the G-protein composition of both apical and basolateral membrane vesicles. Western blots showed an essentially uniform distribution of G alpha total, G alpha S and G beta over the two membranes. Go could not be detected with western blot technique in the vesicle preparations. By contrast, the distribution of ADP-ribosylation with the bacterial toxins pertussis toxin and cholera toxin depended on the amount of detergent in the assay and perhaps other factors, and thus could not be used to evaluate the relative amounts of G-protein subunits. Thus, in contrast to the situation in cultured renal cells, unequal distribution of receptor and G-protein substrates is apparently not paralleled by an unequal distribution of the detected forms of G-proteins under physiological conditions.
Acta Physiologica Scandinavica 06/1991; 142(1):83-90. · 2.55 Impact Factor
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ABSTRACT: Vasoactive intestinal polypeptide (VIP) was biotinyl-epsilon-aminocaproylated using sulfosuccinimidyl-6-(biotinamido) hexanoate thereby producing a series of products that were separated by high performance liquid chromatography (HPLC). Seven VIP-derivatives were isolated and the number and location of biotinyl-epsilon-aminocaproylation was determined by a combination of enzymatic degradation and plasma desorption mass spectrometry (PDMS). Receptor binding experiments with the VIP biotinyl-epsilon-aminocaproylated derivatives revealed IC50 values for the monobiotinyl-epsilon-aminocaproylated peptides that were 1.3-3.2 times higher than for natural VIP. All isolated biotinyl-epsilon-aminocaproylated derivatives possess VIP-like bioactivity as shown by an assay measuring pancreatic juice secretion in cat, VIP biotinyl-epsilon-aminocaproylated in position lysine being almost equipotent with natural VIP.
FEBS Letters 05/1991; 282(1):35-40. · 3.54 Impact Factor
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ABSTRACT: We describe the structure of a gene expressed in the salivary gland cells of the dipteran Chironomus tentans and show that it encodes 1 of the approximately 15 secretory proteins exported by the gland cells. This sp115,140 gene consists of approximately 65 copies of a 42-bp sequence in a central uninterrupted core block, surrounded by short nonrepetitive regions. The repeats within the gene are highly similar to each other, but divergent repeats are present in a pattern which suggests that the repeat structure has been remodeled during evolution. The 42-bp repeat in the gene is a simple variant of the more complex repeat unit present in the Balbiani ring genes, encoding four of the other secretory proteins. The structure of the sp115,140 gene suggests that related repeat structures have evolved from a common origin and resulted in the set of genes whose secretory proteins interact in the assembly of the secreted protein fibers.
Journal of Molecular Evolution 08/1990; 31(1):40-50. · 2.27 Impact Factor
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ABSTRACT: The conformations of the neuropeptide galanin in water and trifluoroethanol solutions have been examined by 1H NMR spectroscopy. Analysis of two-dimensional NMR experiments enabled the assignment of virtually all the 1H resonances of galanin in trifluoroethanol solution and many of the 1H resonances in aqueous solution. Interpretation of the NMR data in structural terms suggests that in trifluoroethanol galanin is predominantly helical while in water it does not adopt a fixed conformation.
Biochemical and Biophysical Research Communications 03/1990; 166(3):1102-9. · 2.48 Impact Factor
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ABSTRACT: The structure of rat galanin, recently elucidated using recombinant DNA technology, differs from porcine galanin by three amino acid residue substitutions at positions located in the C-terminal region. A synthetic replicate of the proposed structure of rat galanin was prepared and its potency to inhibit insulin responses to glucose in anesthetized rats was compared with that of porcine galanin. Within experimental error, the dose-response curves of porcine and rat galanin to inhibit glucose-stimulated rat insulin responses were indistinguishable. The ED50 dose of porcine galanin was 0.6 micrograms/100 g body weight and for rat galanin 0.8 micrograms/100 g body weight. These results suggest that the C-terminal region of the molecule is not essential for galanin's potent inhibitory action on insulin responses to glucose administration.
Pancreas 02/1990; 5(1):70-4. · 2.39 Impact Factor
