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1.
J Dent Res ; 88(9): 851-5, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19767584

ABSTRACT

Periodontal diseases, such as gingivitis and periodontitis, are characterized by bacterial plaque accumulation around the gingival crevice and the subsequent inflammation and destruction of host tissues. To test the hypothesis that cellular metabolism is altered as a result of host-bacteria interaction, we performed an unbiased metabolomic profiling of gingival crevicular fluid (GCF) collected from healthy, gingivitis, and periodontitis sites in humans, by liquid and gas chromatography mass spectrometry. The purine degradation pathway, a major biochemical source for reactive oxygen species (ROS) production, was significantly accelerated at the disease sites. This suggests that periodontal-disease-induced oxidative stress and inflammation are mediated through this pathway. The complex host-bacterial interaction was further highlighted by depletion of anti-oxidants, degradation of host cellular components, and accumulation of bacterial products in GCF. These findings provide new mechanistic insights and a panel of comprehensive biomarkers for periodontal disease progression.


Subject(s)
Periodontal Diseases/metabolism , Purines/metabolism , Adult , Aged , Cadaverine/analysis , Chronic Periodontitis/metabolism , Female , Gingiva/metabolism , Gingival Crevicular Fluid/chemistry , Gingival Crevicular Fluid/metabolism , Gingival Hemorrhage/metabolism , Gingivitis/metabolism , Glutathione/analysis , Host-Pathogen Interactions , Humans , Hypoxanthine/analysis , Male , Metabolome , Middle Aged , Oxidative Stress/physiology , Periodontal Attachment Loss/metabolism , Periodontal Pocket/metabolism , Purines/analysis , Putrescine/analysis , Reactive Oxygen Species/analysis , Up-Regulation , Uric Acid/analysis , Xanthine/analysis
2.
Genes Dev ; 14(17): 2229-41, 2000 Sep 01.
Article in English | MEDLINE | ID: mdl-10970886

ABSTRACT

The 9-cis-retinoic acid receptors (RXRalpha, RXRbeta, and RXRgamma) are nuclear receptors that play key roles in multiple hormone-signaling pathways. Biochemical data indicate that, in the absence of ligand, RXR can exist as an inactive tetramer and that its dissociation, induced by ligand, is important for receptor activation. In this article we report the inactivated tetramer structures of the RXRalpha ligand-binding domain (LBD), either in the absence of or in the presence of a nonactivating ligand. These structures reveal that the RXR LBD tetramer forms a compact, disc-shaped complex, consisting of two symmetric dimers that are packed along helices 3 and 11. In each monomer, the AF-2 helix protrudes away from the core domain and spans into the coactivator binding site in the adjacent monomer of the symmetric dimer. In this configuration, the AF-2 helix physically excludes the binding of coactivators and suggests an autorepression mechanism that is mediated by the AF-2 helix within the tetramer. The RXR-tetramer interface is assembled from amino acids that are conserved across several closely related receptors, including the HNF4s and COUP transcription factors, and may therefore provide a model for understanding structure and regulation of this subfamily of nuclear receptors.


Subject(s)
Receptors, Retinoic Acid/chemistry , Receptors, Retinoic Acid/metabolism , Transcription Factors/chemistry , Transcription Factors/metabolism , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Dimerization , Gene Expression Regulation , Ligands , Models, Molecular , Molecular Sequence Data , Plasmids/chemistry , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Retinoid X Receptors , Sequence Homology, Amino Acid , Stereoisomerism , Tretinoin/chemistry
3.
Mol Cell ; 5(3): 545-55, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10882139

ABSTRACT

The nuclear receptor PPARgamma/RXRalpha heterodimer regulates glucose and lipid homeostasis and is the target for the antidiabetic drugs GI262570 and the thiazolidinediones (TZDs). We report the crystal structures of the PPARgamma and RXRalpha LBDs complexed to the RXR ligand 9-cis-retinoic acid (9cRA), the PPARgamma agonist rosiglitazone or GI262570, and coactivator peptides. The PPARgamma/RXRalpha heterodimer is asymmetric, with each LBD deviated approximately 10 degrees from the C2 symmetry, allowing the PPARgamma AF-2 helix to interact with helices 7 and 10 of RXRalpha. The heterodimer interface is composed of conserved motifs in PPARgamma and RXRalpha that form a coiled coil along helix 10 with additional charge interactions from helices 7 and 9. The structures provide a molecular understanding of the ability of RXR to heterodimerize with many nuclear receptors and of the permissive activation of the PPARgamma/RXRbeta heterodimer by 9cRA.


Subject(s)
Nuclear Proteins/chemistry , Receptors, Cytoplasmic and Nuclear/chemistry , Receptors, Retinoic Acid/chemistry , Thiazolidinediones , Transcription Factors/chemistry , Alitretinoin , Amino Acid Sequence , Binding Sites , Crystallography , Diabetes Mellitus, Type 2/drug therapy , Dimerization , Drug Design , Histone Acetyltransferases , Humans , Ligands , Models, Molecular , Molecular Sequence Data , Nuclear Receptor Coactivator 1 , Receptors, Cytoplasmic and Nuclear/agonists , Retinoid X Receptors , Rosiglitazone , Sequence Homology, Amino Acid , Surface Properties , Thiazoles/chemistry , Transcription Factors/agonists , Tretinoin/chemistry
4.
Science ; 288(5472): 1822-5, 2000 Jun 09.
Article in English | MEDLINE | ID: mdl-10846163

ABSTRACT

Cyclic nucleotides are second messengers that are essential in vision, muscle contraction, neurotransmission, exocytosis, cell growth, and differentiation. These molecules are degraded by a family of enzymes known as phosphodiesterases, which serve a critical function by regulating the intracellular concentration of cyclic nucleotides. We have determined the three-dimensional structure of the catalytic domain of phosphodiesterase 4B2B to 1.77 angstrom resolution. The active site has been identified and contains a cluster of two metal atoms. The structure suggests the mechanism of action and basis for specificity and will provide a framework for structure-assisted drug design for members of the phosphodiesterase family.


Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases/chemistry , 3',5'-Cyclic-AMP Phosphodiesterases/metabolism , Cyclic AMP/metabolism , Binding Sites , Catalytic Domain , Crystallization , Crystallography, X-Ray , Cyclic AMP/chemistry , Cyclic GMP/chemistry , Cyclic GMP/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 4 , Hydrogen Bonding , Hydrolysis , Metals/metabolism , Models, Molecular , Protein Conformation , Protein Folding , Protein Structure, Secondary , Substrate Specificity
5.
Acta Crystallogr D Biol Crystallogr ; 56(Pt 4): 472-4, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10739924

ABSTRACT

Crystals of the Saccharomyces cerevisiae pyrimidine biosynthetic enzyme orotidine 5'-phosphate decarboxylase (ODCase) were grown by the hanging-drop vapor-diffusion technique at 277 K using polyethylene glycol 4000 as the precipitant. Crystals of native and selenomethionyl ODCase diffract to less than 2.2 A and belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 90.1, b = 116.2, c = 117.0 A. Crystals of ODCase grown in the presence of the postulated transition-state analog inhibitor 6-hydroxyuridine 5'--phosphate (BMP) diffract to less than 2.5 A and belong to space group P2(1), with unit-cell parameters a = 79.9, b = 80.0, c = 98.2 A, beta = 108.6 degrees.


Subject(s)
Orotidine-5'-Phosphate Decarboxylase/chemistry , Saccharomyces cerevisiae/enzymology , Crystallization , Crystallography, X-Ray , Escherichia coli , Orotidine-5'-Phosphate Decarboxylase/isolation & purification , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Selenomethionine/metabolism
6.
Proc Natl Acad Sci U S A ; 97(5): 2011-6, 2000 Feb 29.
Article in English | MEDLINE | ID: mdl-10681417

ABSTRACT

Orotidine 5'-phosphate decarboxylase produces the largest rate enhancement that has been reported for any enzyme. The crystal structure of the recombinant Saccharomyces cerevisiae enzyme has been determined in the absence and presence of the proposed transition state analog 6-hydroxyuridine 5'-phosphate, at a resolution of 2.1 A and 2.4 A, respectively. Orotidine 5'-phosphate decarboxylase folds as a TIM-barrel with the ligand binding site near the open end of the barrel. The binding of 6-hydroxyuridine 5'-phosphate is accompanied by protein loop movements that envelop the ligand almost completely, forming numerous favorable interactions with the phosphoryl group, the ribofuranosyl group, and the pyrimidine ring. Lysine-93 appears to be anchored in such a way as to optimize electrostatic interactions with developing negative charge at C-6 of the pyrimidine ring, and to donate the proton that replaces the carboxylate group at C-6 of the product. In addition, H-bonds from the active site to O-2 and O-4 help to delocalize negative charge in the transition state. Interactions between the enzyme and the phosphoribosyl group anchor the pyrimidine within the active site, helping to explain the phosphoribosyl group's remarkably large contribution to catalysis despite its distance from the site of decarboxylation.


Subject(s)
Orotidine-5'-Phosphate Decarboxylase/chemistry , Amino Acid Sequence , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Protein Conformation , Recombinant Fusion Proteins/chemistry , Saccharomyces cerevisiae/enzymology
7.
Recent Prog Horm Res ; 54: 345-67; discussion 367-8, 1999.
Article in English | MEDLINE | ID: mdl-10548883

ABSTRACT

Traditional pharmacologic approaches had identified several classes of xenobiotics that elicited characteristic biological effects in vivo but that lacked defined molecular mechanisms of action. Among these xenobiotics were the peroxisome proliferators, the thiazolidinediones (TZDs), and a set of compounds that induced the expression of cytochrome P450 (CYP) 3A genes and promoted the metabolism of other xenobiotics. All three classes of xenobiotics are now known to exert their actions through activation of orphan members of the nuclear receptor family of ligand-activated transcription factors. Peroxisome proliferators are a diverse group of amphipathic acids that include the fibrate class of triglyceride- and cholesterol-lowering drugs. TZDs sensitize tissues such as skeletal muscle, liver, and adipose to the actions of insulin and lower glucose and lipid levels in type 2 diabetics. The peroxisome proliferators and TZDs are now known to mediate their effects through the peroxisome proliferator-activated receptors (PPARs) alpha and gamma, respectively. The activities of these compounds established the PPARs as key regulators of glucose and lipid homeostasis. We and others have recently shown that various naturally occurring fatty acids and eicosanoids serve as PPAR ligands, suggesting a novel regulatory mechanism whereby dietary lipids and their metabolites can regulate gene transcription and impact overall energy balance. The third class of xenobiotics we have studied induces the expression of CYP3A genes, mono-oxygenases responsible for the metabolism of natural steroids as well as a variety of xenobiotics, including > 60% of all drugs. We have recently shown that compounds that induce CYP3A gene expression do so through activation of novel orphan receptors, termed the pregnane X receptors (PXRs). Many of the PXR activators are widely used drugs such as dexamethasone, lovastatin, and rifampicin, whose induction of CYP3A levels causes them to promote the metabolism of other drugs, often with adverse consequences. Thus, the finding that the PXRs regulate CYP3A gene expression provides a basis for the efficient identification and elimination of candidate drugs that will interact with other medicines. Searches for natural ligands have revealed that the PXRs are activated by C21 steroids, including pregnenolone and progesterone, suggesting that these orphan receptors define a novel steroid hormone signaling pathway. In sum, work from our laboratories and others has demonstrated that peroxisome proliferators, TZDs, and inducers of CYP3A gene expression exert their biological actions through the activation of orphan nuclear receptors. These findings provide insights into new endocrine signaling pathways and have important implications for the discovery of safer and more efficacious drugs for the treatment of a variety of diseases.


Subject(s)
Nuclear Proteins/physiology , Receptors, Cytoplasmic and Nuclear/physiology , Receptors, Steroid/physiology , Signal Transduction/physiology , Transcription Factors/physiology , Xenobiotics/metabolism , Animals , Humans , Pregnane X Receptor , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Steroid/metabolism , Transcription Factors/metabolism
8.
Proc Natl Acad Sci U S A ; 96(11): 6102-6, 1999 May 25.
Article in English | MEDLINE | ID: mdl-10339548

ABSTRACT

The peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that regulate glucose and lipid homeostasis. The PPARgamma subtype plays a central role in the regulation of adipogenesis and is the molecular target for the 2, 4-thiazolidinedione class of antidiabetic drugs. Structural studies have revealed that agonist ligands activate the PPARs through direct interactions with the C-terminal region of the ligand-binding domain, which includes the activation function 2 helix. GW0072 was identified as a high-affinity PPARgamma ligand that was a weak partial agonist of PPARgamma transactivation. X-ray crystallography revealed that GW0072 occupied the ligand-binding pocket by using different epitopes than the known PPAR agonists and did not interact with the activation function 2 helix. In cell culture, GW0072 was a potent antagonist of adipocyte differentiation. These results establish an approach to the design of PPAR ligands with modified biological activities.


Subject(s)
Adipocytes/cytology , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Receptors, Cytoplasmic and Nuclear/chemistry , Thiazoles/pharmacology , Thiazolidinediones , Transcription Factors/antagonists & inhibitors , Transcription Factors/chemistry , Animals , Binding Sites , Cell Differentiation/drug effects , Cell Line , Chlorocebus aethiops , Crystallography, X-Ray , Humans , Kinetics , Ligands , Mice , Models, Molecular , Molecular Conformation , Molecular Sequence Data , Nuclear Proteins/metabolism , Protein Structure, Secondary , Receptors, Cytoplasmic and Nuclear/genetics , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Rosiglitazone , Thiazoles/chemical synthesis , Thiazoles/chemistry , Thiazolidines , Transcription Factors/genetics , Transfection
9.
Mol Cell ; 3(3): 397-403, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10198642

ABSTRACT

The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors for fatty acids (FAs) that regulate glucose and lipid homeostasis. We report the crystal structure of the PPAR delta ligand-binding domain (LBD) bound to either the FA eicosapentaenoic acid (EPA) or the synthetic fibrate GW2433. The carboxylic acids of EPA and GW2433 interact directly with the activation function 2 (AF-2) helix. The hydrophobic tail of EPA adopts two distinct conformations within the large hydrophobic cavity. GW2433 occupies essentially the same space as EPA bound in both conformations. These structures provide molecular insight into the propensity for PPARs to interact with a variety of synthetic and natural compounds, including FAs that vary in both chain length and degree of saturation.


Subject(s)
Fatty Acids/metabolism , Receptors, Cytoplasmic and Nuclear/chemistry , Receptors, Cytoplasmic and Nuclear/metabolism , Transcription Factors/chemistry , Transcription Factors/metabolism , Animals , Binding Sites , Butyrates/chemistry , Butyrates/metabolism , Crystallization , Crystallography, X-Ray , Eicosapentaenoic Acid/chemistry , Eicosapentaenoic Acid/metabolism , Fatty Acids/chemistry , Humans , Hydrogen Bonding , Inhibitory Concentration 50 , Ligands , Models, Molecular , Molecular Conformation , Phenylurea Compounds/chemistry , Phenylurea Compounds/metabolism , Protein Binding , Protein Conformation , Protein Structure, Secondary
10.
Genes Dev ; 13(24): 3198-208, 1999 Dec 15.
Article in English | MEDLINE | ID: mdl-10617569

ABSTRACT

Retinoic acid and thyroid hormone receptors can act alternatively as ligand-independent repressors or ligand-dependent activators, based on an exchange of N-CoR or SMRT-containing corepressor complexes for coactivator complexes in response to ligands. We provide evidence that the molecular basis of N-CoR recruitment is similar to that of coactivator recruitment, involving cooperative binding of two helical interaction motifs within the N-CoR carboxyl terminus to both subunits of a RAR-RXR heterodimer. The N-CoR and SMRT nuclear receptor interaction motifs exhibit a consensus sequence of LXX I/H I XXX I/L, representing an extended helix compared to the coactivator LXXLL helix, which is able to interact with specific residues in the same receptor pocket required for coactivator binding. We propose a model in which discrimination of the different lengths of the coactivator and corepressor interaction helices by the nuclear receptor AF2 motif provides the molecular basis for the exchange of coactivators for corepressors, with ligand-dependent formation of the charge clamp that stabilizes LXXLL binding sterically inhibiting interaction of the extended corepressor helix.


Subject(s)
Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Receptors, Retinoic Acid/metabolism , Receptors, Thyroid Hormone/metabolism , Repressor Proteins/chemistry , Repressor Proteins/metabolism , Amino Acid Sequence , Animals , Cell Line , Consensus Sequence , Conserved Sequence , Dimerization , HeLa Cells , Humans , Macromolecular Substances , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Nuclear Proteins/genetics , Nuclear Receptor Co-Repressor 1 , Protein Structure, Secondary , Rats , Receptors, Retinoic Acid/chemistry , Receptors, Thyroid Hormone/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Repressor Proteins/genetics , Retinoid X Receptors , Sequence Alignment , Sequence Homology, Amino Acid , Transcription Factors/chemistry , Transcription Factors/metabolism
11.
Genes Dev ; 12(21): 3357-68, 1998 Nov 01.
Article in English | MEDLINE | ID: mdl-9808623

ABSTRACT

Ligand-dependent activation of gene transcription by nuclear receptors is dependent on the recruitment of coactivators, including a family of related NCoA/SRC factors, via a region containing three helical domains sharing an LXXLL core consensus sequence, referred to as LXDs. In this manuscript, we report receptor-specific differential utilization of LXXLL-containing motifs of the NCoA-1/SRC-1 coactivator. Whereas a single LXD is sufficient for activation by the estrogen receptor, different combinations of two, appropriately spaced, LXDs are required for actions of the thyroid hormone, retinoic acid, peroxisome proliferator-activated, or progesterone receptors. The specificity of LXD usage in the cell appears to be dictated, at least in part, by specific amino acids carboxy-terminal to the core LXXLL motif that may make differential contacts with helices 1 and 3 (or 3') in receptor ligand-binding domains. Intriguingly, distinct carboxy-terminal amino acids are required for PPARgamma activation in response to different ligands. Related LXXLL-containing motifs in NCoA-1/SRC-1 are also required for a functional interaction with CBP, potentially interacting with a hydrophobic binding pocket. Together, these data suggest that the LXXLL-containing motifs have evolved to serve overlapping roles that are likely to permit both receptor-specific and ligand-specific assembly of a coactivator complex, and that these recognition motifs underlie the recruitment of coactivator complexes required for nuclear receptor function.


Subject(s)
Peptide Fragments/chemistry , Receptors, Cytoplasmic and Nuclear/chemistry , Transcription Factors/chemistry , Transcriptional Activation/physiology , Amino Acid Sequence , Animals , Cells, Cultured , Fibroblasts/cytology , Gene Expression Regulation , Histone Acetyltransferases , Models, Molecular , Molecular Sequence Data , Nuclear Proteins/physiology , Nuclear Receptor Coactivator 1 , Peptide Fragments/genetics , Peptide Fragments/physiology , Protein Structure, Secondary , Rats , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Steroid/chemistry , Receptors, Steroid/genetics , Sequence Alignment , Trans-Activators/physiology , Transcription Factors/genetics
12.
Nature ; 395(6698): 137-43, 1998 Sep 10.
Article in English | MEDLINE | ID: mdl-9744270

ABSTRACT

The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a ligand-dependent transcription factor that is important in adipocyte differentiation and glucose homeostasis and which depends on interactions with co-activators, including steroid receptor co-activating factor-1 (SRC-1). Here we present the X-ray crystal structure of the human apo-PPAR-gamma ligand-binding domain (LBD), at 2.2 A resolution; this structure reveals a large binding pocket, which may explain the diversity of ligands for PPAR-gamma. We also describe the ternary complex containing the PPAR-gamma LBD, the antidiabetic ligand rosiglitazone (BRL49653), and 88 amino acids of human SRC-1 at 2.3 A resolution. Glutamate and lysine residues that are highly conserved in LBDs of nuclear receptors form a 'charge clamp' that contacts backbone atoms of the LXXLL helices of SRC-1. These results, together with the observation that two consecutive LXXLL motifs of SRC-1 make identical contacts with both subunits of a PPAR-gamma homodimer, suggest a general mechanism for the assembly of nuclear receptors with co-activators.


Subject(s)
Receptors, Cytoplasmic and Nuclear/chemistry , Thiazolidinediones , Transcription Factors/chemistry , Amino Acid Sequence , Binding Sites , Cloning, Molecular , Conserved Sequence , Crystallography, X-Ray , Escherichia coli , Histone Acetyltransferases , Humans , Hypoglycemic Agents/chemistry , Hypoglycemic Agents/metabolism , Ligands , Macromolecular Substances , Molecular Sequence Data , Nuclear Receptor Coactivator 1 , Protein Conformation , Protein Structure, Tertiary , Receptors, Cytoplasmic and Nuclear/metabolism , Rosiglitazone , Structure-Activity Relationship , Thiazoles/chemistry , Thiazoles/metabolism , Transcription Factors/metabolism , Transcriptional Activation
13.
Nature ; 395(6698): 199-202, 1998 Sep 10.
Article in English | MEDLINE | ID: mdl-9744281

ABSTRACT

Retinoic-acid receptor-alpha (RAR-alpha) and peroxisome proliferator-activated receptor-gamma (PPAR-gamma) are members of the nuclear-receptor superfamily that bind to DNA as heterodimers with retinoid-X receptors (RXRs). PPAR-RXR heterodimers can be activated by PPAR or RXR ligands, whereas RAR-RXR heterodimers are selectively activated by RAR ligands only, because of allosteric inhibition of the binding of ligands to RXR by RAR. However, RXR ligands can potentiate the transcriptional effects of RAR ligands in cells. Transcriptional activation by nuclear receptors requires a carboxy-terminal helical region, termed activation function-2 (AF-2), that forms part of the ligand-binding pocket and undergoes a conformational change required for the recruitment of co-activator proteins, including NCoA-1/SRC-1. Here we show that allosteric inhibition of RXR results from a rotation of the RXR AF-2 helix that places it in contact with the RAR coactivator-binding site. Recruitment of an LXXLL motif of SRC-1 to RAR in response to ligand displaces the RXR AF-2 domain, allowing RXR ligands to bind and promote the binding of a second LXXLL motif from the same SRC-1 molecule. These results may partly explain the different responses of nuclear-receptor heterodimers to RXR-specific ligands.


Subject(s)
Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Retinoic Acid/metabolism , Transcription Factors/metabolism , Allosteric Regulation , Amino Acid Sequence , Benzoates/metabolism , Binding Sites , Cell Line , Histone Acetyltransferases , Ligands , Models, Molecular , Molecular Sequence Data , Nuclear Receptor Coactivator 1 , Protein Conformation , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Retinoic Acid/antagonists & inhibitors , Receptors, Retinoic Acid/genetics , Recombinant Fusion Proteins/metabolism , Retinoid X Receptors , Retinoids/metabolism , Transcription Factors/genetics
15.
J Mol Biol ; 236(5): 1410-2, 1994 Mar 11.
Article in English | MEDLINE | ID: mdl-8126730

ABSTRACT

Crystals of the catalytic domain of human fibroblast collagenase have been grown in the presence and absence of an inhibitor. Crystals of the inhibitor complex grew from 0.2 M ammonium sulfate and 15 to 30% PEG 8000 at 22 degrees C as bipyramids in the space group P6(2) or P6(4). Crystals of the unligated enzyme grew as rods in the space group P4(1)2(1)2 or P4(3)2(1)2 from 1.0 to 2.0 M sodium formate at 4 degrees C. Both crystal forms grew quite slowly over a period of months, but ultimately yielded crystals that diffracted beyond 2.5 A. The collagenase samples used in these studies were heterogeneous at the amino terminus. Three major species (full length, N-1 and N-2) were identified by mass spectrometry and Edman sequencing. Analysis of dissolved crystals revealed the native crystal form selectively crystallized as the N-2 species; however, no selectivity of N-terminal forms was observed for crystals of the inhibitor complex.


Subject(s)
Collagenases/ultrastructure , Crystallography, X-Ray , Fibroblasts/enzymology , Humans , Macromolecular Substances , Mass Spectrometry , Matrix Metalloproteinase Inhibitors , Molecular Weight , Recombinant Proteins
16.
J Mol Biol ; 232(2): 555-73, 1993 Jul 20.
Article in English | MEDLINE | ID: mdl-8345523

ABSTRACT

The aspartate receptor is a transmembrane-signalling protein that mediates chemotaxis behaviour in bacteria. Aspartate receptors in Salmonella typhimurium and Escherichia coli exist as dimers of two subunits in the presence as well as in the absence of aspartate. We have previously reported the three-dimensional structures of the external ligand-binding domain of the S. typhimurium aspartate receptor with and without bound aspartate. The external or periplasmic region of the aspartate receptor is a dimer of four-alpha-helical bundle subunits; a single aspartate molecule binds to one of two sites residing at the subunit interface, increasing the affinity of the subunits for one another. Here we report the results of a detailed analysis of the aspartate receptor ligand-binding domain structure (residues 25 to 188). The dimer interface between the twofold related subunits consists primarily of contacts mediated by the side-chains of the N-terminal helix of each four-alpha-helical bundle subunit. The N-terminal helices pack approximately 20 degrees from parallel as an approximate coiled-coil super-secondary structure. We have refined aspartate receptor ligand-binding domain structures in the presence and in the absence of a bound aromatic compound, 1,10-phenanthroline, to 2.2 A and 2.3 A resolution, respectively, as well as crystal structures in the presence of specifically bound Au(I), Hg(II) and Pt(IV) complex ions at 2.4 A, 3.0 A and 3.3 A resolution, respectively. The possible biological relevance of the aromatic ligand-binding site and the metal ion-binding sites is discussed. The dimer of four-alpha-helical bundle subunits composing the periplasmic region of the S. typhimurium aspartate receptor provides a basis for understanding the results of mutational analyses performed on related chemotaxis transmembrane receptors. The crystal structure analysis provides an explanation for the way in which mutations in the E. coli aspartate receptor affect its binding to the periplasmic maltose-binding protein and how mutations in the more distantly related E. coli Trg chemotaxis receptor affect its binding to the periplasmic ribose and glucose-galactose binding proteins.


Subject(s)
Aspartic Acid/metabolism , Bacterial Proteins/chemistry , Escherichia coli Proteins , Peptide Fragments/chemistry , Receptors, Amino Acid/chemistry , Salmonella typhimurium/chemistry , Chemotaxis , Escherichia coli/chemistry , Gold/chemistry , Gold/metabolism , Membrane Proteins/chemistry , Mercury/chemistry , Mercury/metabolism , Models, Molecular , Phenanthrolines/chemistry , Phenanthrolines/metabolism , Platinum/chemistry , Platinum/metabolism , Protein Conformation , Signal Transduction , X-Ray Diffraction
17.
Nature ; 363(6425): 172-6, 1993 May 13.
Article in English | MEDLINE | ID: mdl-8483502

ABSTRACT

Interleukin-5 (IL-5) is a lineage-specific cytokine for eosinophilpoiesis and plays an important part in diseases associated with increased eosinophils, such as asthma. Human IL-5 is a disulphide-linked homodimer with 115 amino-acid residues in each chain. The crystal structure at 2.4 A resolution reveals a novel two-domain structure, with each domain showing a striking similarity to the cytokine fold found in granulocyte macrophage and macrophage colony-stimulating factors, IL-2 (ref. 5), IL-4 (ref. 6), and human and porcine growth hormones. IL-5 is unique in that each domain requires the participation of two chains. The IL-5 structure consists of two left-handed bundles of four helices laid end to end and two short beta-sheets on opposite sides of the molecule. Surprisingly, the C-terminal strand and helix of one chain complete a bundle of four helices and a beta-sheet with the N-terminal three helices and one strand of the other chain. The structure of IL-5 provides a molecular basis for the design of antagonists and agonists that would delineate receptor recognition determinants critical in signal transduction. This structure determination extends the family of the cytokine bundle of four helices and emphasizes its fundamental significance and versatility in recognizing its receptor.


Subject(s)
Interleukin-5/chemistry , Animals , Binding Sites , Computer Simulation , Cytokines/chemistry , Humans , Mice , Models, Molecular , Protein Conformation , Protein Structure, Secondary , Sequence Alignment , X-Ray Diffraction
18.
Eur J Biochem ; 212(3): 751-5, 1993 Mar 15.
Article in English | MEDLINE | ID: mdl-8462547

ABSTRACT

Interleukin-5 (IL-5) plays a key role in the proliferation and differentiation of eosinophils. To aid the solution of the crystallographic three-dimensional structure, we have expressed large quantities of recombinant human IL-5 (hIL-5) in a methionine auxotroph strain of Escherichia coli (DL41) grown on an enriched seleno-DL-methionine-containing medium. Cell densities of A650 = 10 have been achieved. The selenomethionyl-labelled hIL-5 (Se-hIL-5) has been purified and found to contain 3.6 selenium atoms/dimer, and 0.4 methionine residues/dimer. In a B-cell growth factor assay, the Se-hIL-5 is significantly more active than the non-labelled hIL-5. Electrospray mass spectrometry shows two major peaks, with relative molecular masses of 26,326 +/- 6 and 26,280 +/- 8 corresponding to the 4Se and 3Se/1S forms of hIL-5. Unlike the methionine-containing hIL-5, the N-terminal selenomethionine is neither oxidised nor carbamoylated and can only be resolved into two species in isoelectric focusing gel electrophoresis. Se-hIL-5 crystallises in the same space group and unit cell as hIL-5. Difference Fourier calculations identify two of the selenomethionines corresponding to Met107 in the dimer. However, the N-terminal is disordered in the crystal, and the N-terminal selenomethionines are not resolved in the difference Fourier.


Subject(s)
Interleukin-5/analogs & derivatives , Interleukin-5/isolation & purification , Recombinant Proteins/isolation & purification , Selenomethionine/analogs & derivatives , Animals , B-Lymphocytes , Cell Division/drug effects , Chromatography, Gel , Chromatography, Ion Exchange , Crystallization , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Humans , Interleukin-5/chemistry , Interleukin-5/pharmacology , Mice , Recombinant Proteins/chemistry , Recombinant Proteins/pharmacology , Selenomethionine/chemistry , Selenomethionine/isolation & purification , Selenomethionine/pharmacology
19.
J Mol Biol ; 229(4): 1150-2, 1993 Feb 20.
Article in English | MEDLINE | ID: mdl-8445640

ABSTRACT

Recombinant human interleukin-5 (rhIL-5) has been crystallized by the hanging drop vapor diffusion method using 0.1 M-Tris.HCl buffer (pH 8.5) containing 0.2 to 0.25 M-sodium acetate and 26 to 30% PEG 4000 at 22 degrees C. The parallel-piped crystals belong to the space group C2 with unit cell dimensions of a = 122.1 A, b = 36.11 A, c = 56.42 A, beta = 98.59 degrees. They diffract to at least 2.0 A resolution on a rotating anode X-ray source. The molecular mass weight of the protein and the volume of the unit cell suggest that the asymmetric unit contains one intermolecular disulfide-bonded homodimer.


Subject(s)
Interleukin-5/chemistry , Crystallization , Escherichia coli , Humans , Mass Spectrometry , Recombinant Proteins/chemistry , X-Ray Diffraction
20.
Proc Natl Acad Sci U S A ; 89(8): 3649-53, 1992 Apr 15.
Article in English | MEDLINE | ID: mdl-1565661

ABSTRACT

RAS genes isolated from human tumors often have mutations at positions corresponding to amino acid 12 or 61 of the encoded protein (p21), while retroviral ras-encoded p21 contains substitutions at both positions 12 and 59. These mutant proteins are deficient in their GTP hydrolysis activity, and this loss of activity is linked to their transforming potential. The crystal structures of the mutant proteins are presented here as either GDP-bound or GTP-analogue-bound complexes. Based on these structures, a mechanism for the p21 GTPase reaction is proposed that is consistent with the observed structural and biochemical data. The central feature of this mechanism is a specific stabilization complex formed between the Gln-61 side-chain and the pentavalent gamma-phosphate of the GTP transition state. Amino acids other than glutamine at position 61 cannot stabilize the transition state, and amino acids larger than glycine at position 12 would interfere with the transition-state complex. Thr-59 disrupts the normal position of residue 61, thus preventing its participation in the transition-state complex.


Subject(s)
Guanosine Triphosphate/metabolism , Mutation , Proto-Oncogene Proteins p21(ras)/metabolism , Amino Acid Sequence , Binding Sites , Humans , Hydrolysis , Models, Molecular , Molecular Sequence Data , Protein Conformation , Proto-Oncogene Proteins p21(ras)/chemistry , Proto-Oncogene Proteins p21(ras)/genetics , X-Ray Diffraction/methods
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