ABSTRACT
Translationally controlled tumor protein (TCTP) is a potential target for cancer therapy. It functions as a growth regulating protein implicated in the TSC1-TSC2 -mTOR pathway or a guanine nucleotide dissociation inhibitor for the elongation factors EF1A and EF1Bbeta. Accumulating evidence indicates that TCTP also functions as an antiapoptotic protein, through a hitherto unknown mechanism. In keeping with this, we show here that loss of tctp expression in mice leads to increased spontaneous apoptosis during embryogenesis and causes lethality between E6.5 and E9.5. To gain further mechanistic insights into this apoptotic function, we solved and refined the crystal structure of human TCTP at 2.0 A resolution. We found a structural similarity between the H2-H3 helices of TCTP and the H5-H6 helices of Bax, which have been previously implicated in regulating the mitochondrial membrane permeability during apoptosis. By site-directed mutagenesis we establish the relevance of the H2-H3 helices in TCTP's antiapoptotic function. Finally, we show that TCTP antagonizes apoptosis by inserting into the mitochondrial membrane and inhibiting Bax dimerization. Together, these data therefore further confirm the antiapoptotic role of TCTP in vivo and provide new mechanistic insights into this key function of TCTP.
Subject(s)
Apoptosis , Biomarkers, Tumor/chemistry , Biomarkers, Tumor/metabolism , Mitochondria/metabolism , bcl-2-Associated X Protein/metabolism , Amino Acid Sequence , Animals , Biomarkers, Tumor/genetics , Cell Line , Crystallography, X-Ray , Dimerization , Embryonic Development , Humans , Mice , Mice, Knockout , Molecular Sequence Data , Mutation , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Alignment , Tumor Protein, Translationally-Controlled 1 , bcl-2-Associated X Protein/chemistryABSTRACT
The first subatomic resolution structure of a 36 kDa protein [aldose reductase (AR)] is presented. AR was cocrystallized at pH 5.0 with its cofactor NADP+ and inhibitor IDD 594, a therapeutic candidate for the treatment of diabetic complications. X-ray diffraction data were collected up to 0.62 A resolution and treated up to 0.66 A resolution. Anisotropic refinement followed by a blocked matrix inversion produced low standard deviations (<0.005 A). The model was very well ordered overall (CA atoms' mean B factor is 5.5 A2). The model and the electron-density maps revealed fine features, such as H-atoms, bond densities, and significant deviations from standard stereochemistry. Other features, such as networks of hydrogen bonds (H bonds), a large number of multiple conformations, and solvent structure were also better defined. Most of the atoms in the active site region were extremely well ordered (mean B approximately 3 A2), leading to the identification of the protonation states of the residues involved in catalysis. The electrostatic interactions of the inhibitor's charged carboxylate head with the catalytic residues and the charged coenzyme NADP+ explained the inhibitor's noncompetitive character. Furthermore, a short contact involving the IDD 594 bromine atom explained the selectivity profile of the inhibitor, important feature to avoid toxic effects. The presented structure and the details revealed are instrumental for better understanding of the inhibition mechanism of AR by IDD 594, and hence, for the rational drug design of future inhibitors. This work demonstrates the capabilities of subatomic resolution experiments and stimulates further developments of methods allowing the use of the full potential of these experiments.
Subject(s)
Acetates/chemistry , Aldehyde Reductase/chemistry , Enzyme Inhibitors/chemistry , Models, Molecular , Thiocarbamates/chemistry , Acetates/metabolism , Aldehyde Reductase/metabolism , Binding Sites , Crystallography, X-Ray , Drug Design , Electrons , Enzyme Inhibitors/metabolism , Hydrogen/chemistry , Molecular Structure , Protein Conformation , Solvents/chemistry , Thioamides , Thiocarbamates/metabolismABSTRACT
The 43 kDa ATPase domain of Thermus thermophilus gyrase B was overproduced in Escherichia coli and a three-step purification protocol yielded large quantities of highly purified enzyme which remained stable for weeks. Crystals of the 43 kDa domain in complex with novobiocin, one of the most potent inhibitors of bacterial topoisomerases, were obtained. Crystals obtained in the presence of PEG 8000 do not diffract, but a different crystal form was obtained using sodium formate as a precipitating agent. The plate-shaped crystals, which were less than 10 microm in thickness, could be cryocooled directly from the mother liquor and a full diffraction data set was collected to 2.3 A allowing the determination of the first structure of a gyrase B 43K domain in complex with a coumarin.
Subject(s)
DNA Gyrase/chemistry , Thermus thermophilus/enzymology , Adenosine Triphosphatases/chemistry , Crystallization , Crystallography, X-Ray , DNA Gyrase/genetics , DNA Gyrase/isolation & purification , Macromolecular Substances , Molecular Weight , Novobiocin/chemistry , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Thermus thermophilus/geneticsABSTRACT
Aminoacyl-tRNA synthetases play a central role in maintaining accuracy during the translation of the genetic code. To achieve this challenging task they have to discriminate against amino acids that are very closely related not only in structure but also in chemical nature. A 'double-sieve' editing model was proposed in the late seventies to explain how two closely related amino acids may be discriminated. However, a clear understanding of this mechanism required structural information on synthetases that are faced with such a problem of amino acid discrimination. The first structural basis for the editing model came recently from the crystal structure of isoleucyl-tRNA synthetase, a class I synthetase, which has to discriminate against valine. The structure showed the presence of two catalytic sites in the same enzyme, one for activation, a coarse sieve which binds both isoleucine and valine, and another for editing, a fine sieve which binds only valine and rejects isoleucine. Another structure of the enzyme in complex with tRNA showed that the tRNA is responsible for the translocation of the misactivated amino-acid substrate from the catalytic site to the editing site. These studies were mainly focused on class I synthetases and the situation was not clear about how class II enzymes discriminate against similar amino acids. The recent structural and enzymatic studies on threonyl-tRNA synthetase, a class II enzyme, reveal how this challenging task is achieved by using a unique zinc ion in the active site as well as by employing a separate domain for specific editing activity. These studies led us to propose a model which emphasizes the mirror symmetrical approach of the two classes of enzymes and highlights that tRNA is the key player in the evolution of these class of enzymes.
Subject(s)
Amino Acyl-tRNA Synthetases/chemistry , Amino Acyl-tRNA Synthetases/metabolism , Genetic Code , Protein Biosynthesis , Amino Acids/chemistry , Amino Acids/metabolism , Catalytic Domain , Isoleucine-tRNA Ligase/chemistry , Isoleucine-tRNA Ligase/metabolism , Models, Biological , Models, Molecular , Protein Conformation , RNA Editing , Threonine-tRNA Ligase/chemistry , Threonine-tRNA Ligase/metabolismABSTRACT
The retinoic acid-related orphan receptor beta (RORbeta) exhibits a highly restricted neuronal-specific expression pattern in brain, retina and pineal gland. So far, neither a natural RORbeta target gene nor a functional ligand have been identified, and the physiological role of the receptor is not well understood. We present the crystal structure of the ligand-binding domain (LBD) of RORbeta containing a bound stearate ligand and complexed with a coactivator peptide. In the crystal, the monomeric LBD adopts the canonical agonist-bound form. The fatty acid ligand-coactivator peptide combined action stabilizes the transcriptionally active conformation. The large ligand-binding pocket is strictly hydrophobic on the AF-2 side and more polar on the beta-sheet side where the carboxylate group of the ligand binds. Site-directed mutagenesis experiments validate the significance of the present structure. Homology modeling of the other isotypes will help to design isotype-selective agonists and antagonists that can be used to characterize the physiological functions of RORs. In addition, our crystallization strategy can be extended to other orphan nuclear receptors, providing a powerful tool to delineate their functions.
Subject(s)
Models, Molecular , Peptide Fragments/chemistry , Receptors, Cell Surface/chemistry , Receptors, Cytoplasmic and Nuclear , Stearic Acids/chemistry , Transcription Factors/chemistry , Amino Acid Sequence , Animals , Binding Sites/physiology , Crystallography, X-Ray , Histone Acetyltransferases , Ligands , Macromolecular Substances , Molecular Sequence Data , Mutagenesis, Site-Directed , Nuclear Receptor Coactivator 1 , Nuclear Receptor Subfamily 1, Group F, Member 2 , Peptide Fragments/metabolism , Protein Conformation , Protein Structure, Tertiary/physiology , Rats , Receptors, Cell Surface/physiology , Sequence Alignment , Sequence Homology, Amino Acid , Structure-Activity RelationshipABSTRACT
FMOC-L-Leucine (F-L-Leu) is a chemically distinct PPARgamma ligand. Two molecules of F-L-Leu bind to the ligand binding domain of a single PPARgamma molecule, making its mode of receptor interaction distinct from that of other nuclear receptor ligands. F-L-Leu induces a particular allosteric configuration of PPARgamma, resulting in differential cofactor recruitment and translating in distinct pharmacological properties. F-L-Leu activates PPARgamma with a lower potency, but a similar maximal efficacy, than rosiglitazone. The particular PPARgamma configuration induced by F-L-Leu leads to a modified pattern of target gene activation. F-L-Leu improves insulin sensitivity in normal, diet-induced glucose-intolerant, and in diabetic db/db mice, yet it has a lower adipogenic activity. These biological effects suggest that F-L-Leu is a selective PPARgamma modulator that activates some (insulin sensitization), but not all (adipogenesis), PPARgamma-signaling pathways.
Subject(s)
Adipocytes/physiology , Amino Acids/pharmacology , Fluorenes/pharmacology , Leucine/chemistry , Receptors, Cytoplasmic and Nuclear/metabolism , Thiazolidinediones , Transcription Factors/metabolism , Adipocytes/drug effects , Amino Acids/chemistry , Amino Acids/metabolism , Animals , Binding Sites , Blood Glucose/metabolism , Body Weight , Cell Differentiation , Cell Line , Dose-Response Relationship, Drug , Fluorenes/chemistry , Fluorenes/metabolism , Gene Expression Regulation/physiology , Genes, Reporter , Hypoglycemic Agents/pharmacology , Insulin Resistance/physiology , Leucine/metabolism , Ligands , Male , Mice , Mice, Inbred Strains , Molecular Structure , Protein Binding , Protein Conformation , Receptors, Cytoplasmic and Nuclear/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Rosiglitazone , Spectrometry, Mass, Electrospray Ionization , Thiazoles/pharmacology , Transcription Factors/genetics , Transcriptional Activation , Tyrosine/chemistry , Tyrosine/metabolismABSTRACT
The 2.6 A resolution crystal structure of an inactive complex between yeast tRNA(Asp) and Escherichia coli aspartyl-tRNA synthetase reveals the molecular details of a tRNA-induced mechanism that controls the specificity of the reaction. The dimer is asymmetric, with only one of the two bound tRNAs entering the active site cleft of its subunit. However, the flipping loop, which controls the proper positioning of the amino acid substrate, acts as a lid and prevents the correct positioning of the terminal adenosine. The structure suggests that the acceptor stem regulates the loop movement through sugar phosphate backbone- protein interactions. Solution and cellular studies on mutant tRNAs confirm the crucial role of the tRNA three-dimensional structure versus a specific recognition of bases in the control mechanism.
Subject(s)
Aspartate-tRNA Ligase/chemistry , RNA, Transfer, Asp/chemistry , RNA, Transfer, Asp/physiology , Base Sequence , Binding Sites , Cell Division , Crystallography, X-Ray , Escherichia coli/cytology , Escherichia coli/enzymology , Escherichia coli/genetics , Gene Deletion , Hydrogen Bonding , Macromolecular Substances , Models, Molecular , Molecular Sequence Data , Protein Subunits , RNA, Transfer, Asp/genetics , Saccharomyces cerevisiae/genetics , Species SpecificityABSTRACT
Several crystal structures of human estrogen receptor alpha ligand-binding domain (hERalpha LBD) complexed with agonist or antagonist molecules have previously been solved. The proteins had been modified in cysteine residues (carboxymethylation) or renatured in urea to circumvent aggregation and denaturation problems. In this work, high-level protein expression and purification together with crystallization screening procedure yielded high amounts of soluble protein without renaturation or modifications steps. The native protein crystallizes in the space group P3(2) 21 with three molecules in the asymmetric unit. The overall structure is very similar to that previously reported for the hERalpha LBD with cysteine carboxymethylated residues thus validating the modification approach. The present strategy can be adapted to other cases where the solubility and the proper folding is a difficulty.
Subject(s)
Peptide Fragments/chemistry , Peptide Fragments/isolation & purification , Receptors, Estrogen/chemistry , Receptors, Estrogen/isolation & purification , Cloning, Molecular , Computer Simulation , Crystallization , Crystallography, X-Ray , Dimerization , Estrogen Receptor alpha , Humans , Ligands , Models, Molecular , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Folding , Protein Structure, Secondary/genetics , Protein Structure, Tertiary/genetics , Receptors, Estrogen/genetics , Receptors, Estrogen/metabolism , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolismSubject(s)
Receptors, Cytoplasmic and Nuclear/chemistry , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Steroid/chemistry , Receptors, Steroid/metabolism , Amino Acid Sequence , Animals , Binding Sites , Humans , Models, Molecular , Molecular Sequence Data , Protein Conformation , Receptors, Cytoplasmic and Nuclear/agonists , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Receptors, Steroid/agonists , Receptors, Steroid/antagonists & inhibitors , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
Nuclear receptors (NRs) form a superfamily of ligand-inducible transcription factors composed of several domains. Recent structural studies focused on domain E, which harbors the ligand-binding site and the ligand-dependent transcription activation function AF-2. Structures of single representatives in an increasing number of various complexes as well as new structures of further NRs addressed issues such as discrimination of ligands, superagonism, isotype specificity, and partial agonism. Until today, one unique transcriptionally active form of domain E was determined; however, divergent tertiary structures of apo-forms and transcriptionally inactive forms are known. Thus, recent results link the transformation of NRs upon ligand binding to principles of protein folding. Furthermore, the ensemble of NR structures, including those of DNA-binding domains, provides one of the foundations for the understanding of interactions with transcription intermediary factors up to the characterization of the link between NR complexes and the basal transcriptional machinery at the structural level.
Subject(s)
Cell Nucleus/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Cytoplasmic and Nuclear/physiology , Transcription, Genetic , Animals , DNA/metabolism , Humans , Ligands , Models, Biological , Models, Chemical , Protein Binding , Protein Folding , Protein Structure, TertiaryABSTRACT
The crystal structures of the ligand-binding domain (LBD) of the vitamin D receptor complexed to 1alpha,25(OH)(2)D(3) and the 20-epi analogs, MC1288 and KH1060, show that the protein conformation is identical, conferring a general character to the observation first made for retinoic acid receptor (RAR) that, for a given LBD, the agonist conformation is unique, the ligands adapting to the binding pocket. In all complexes, the A- to D-ring moieties of the ligands adopt the same conformation and form identical contacts with the protein. Differences are observed only for the 17beta-aliphatic chains that adapt their conformation to anchor the 25-hydroxyl group to His-305 and His-397. The inverted geometry of the C20 methyl group induces different paths of the aliphatic chains. The ligands exhibit a low-energy conformation for MC1288 and a more strained conformation for the two others. KH1060 compensates this energy cost by additional contacts. Based on the present data, the explanation of the superagonist effect is to be found in higher stability and longer half-life of the active complex, thereby excluding different conformations of the ligand binding domain.
Subject(s)
Receptors, Calcitriol/chemistry , Calcitriol/chemistry , Calcitriol/metabolism , Crystallography, X-Ray , Humans , Ligands , Models, Molecular , Protein Conformation , Receptors, Calcitriol/agonists , Receptors, Calcitriol/metabolismABSTRACT
In higher eukaryotes, vitamin A derived metabolites such as 9-cis and all-trans retinoic acid (RA), are involved in the regulation of several essential physiological processes. Their pleiotropic physiological effects are mediated through direct binding to cognate nuclear receptors RXRs and RARs that act as regulated transcription factors belonging to the superfamily of nuclear hormone receptors. Hormone binding to the structurally conserved ligand-binding domain (LBD) of these receptors triggers a conformational change that principally affects the conserved C-terminal transactivation helix H12 involved in transcriptional activation. We report an extensive biophysical solution study of RAR alpha, RXR alpha LBDs and their corresponding RXR alpha/RAR alpha LBD heterodimers combining analytical ultracentrifugation (AUC), small-angle X-ray and neutron scattering (SAXS and SANS) and ab initio three-dimensional shape reconstruction at low resolution. We show that the crystal structures of RXRs and RARs LBDs correlate well with the average conformations observed in solution. Furthermore we demonstrate the effects of 9-cisRA and all-transRA binding on the association properties and conformations of RXR alpha and RAR alpha LBDs in solution. The present study shows that in solution RAR alpha LBD behaves as a monomer in both unliganded and liganded forms. It confirms the existence in solution of a ligand-induced conformational change towards a more compact form of the LBD. It also confirms the stability of the predicted RXR alpha/RAR alpha LBD heterodimers in solution. SAS measurements performed on three different types of RXR alpha/RAR alpha LBD heterodimers (apo/apo, apo/holo and holo/holo) with respect to their ligand-binding site occupancy show the existence of three conformational states depending on the progressive binding of RA stereoisomers on RAR alpha and RXR alpha LBD subunits in the heterodimeric context. These results suggest that the subunits are structurally independent within the heterodimers. Our study also underlines the particular behaviour of RXR alpha LBD. In solution unliganded RXR alpha LBD is observed as two species that are unambiguously identified as homotetramers and homodimers. Molecular modelling combined with SAS data analysis allows us to propose a structural model for this autorepressed apo-tetramer. In contrast to the monomeric state observed in the crystal structure, our data show that in solution active holo-RXR alpha LBD bound to 9-cisRA is a homodimer regardless of the protein concentration. This study demonstrates the crucial role of ligands in the regulation of homodimeric versus heterodimeric association state of RXR in the NR signalling pathways.
Subject(s)
Receptors, Retinoic Acid/metabolism , Transcription Factors/metabolism , Tretinoin/metabolism , Apoproteins/metabolism , Binding Sites , Computer Simulation , Crystallography/methods , Dimerization , Humans , Ligands , Models, Molecular , Mutation , Neutrons , Protein Binding , Protein Conformation , Protein Structure, Quaternary , Protein Structure, Tertiary , Receptors, Retinoic Acid/chemistry , Receptors, Retinoic Acid/genetics , Retinoid X Receptors , Scattering, Radiation , Solutions , Stereoisomerism , Transcription Factors/chemistry , Transcription Factors/genetics , Transcriptional Activation , Ultracentrifugation , X-RaysABSTRACT
Stromelysin-3 (ST3) is a matrix metalloproteinase (MMP-11) whose proteolytic activity plays an important role in tumorigenicity enhancement. In breast cancer, ST3 is a bad prognosis marker: its expression is associated with a poor clinical outcome. This enzyme therefore represents an attractive therapeutic target. The topology of matrix metalloproteinases (MMPs) is remarkably well conserved, making the design of highly specific inhibitors difficult. The major difference between MMPs lies in the S(1)' subsite, a well-defined hydrophobic pocket of variable depth. The present crystal structure, the first 3D-structure of the ST3 catalytic domain in interaction with a phosphinic inhibitor mimicking a (d, l) peptide, clearly demonstrates that its S(1)' pocket corresponds to a tunnel running through the enzyme. This open channel is filled by the inhibitor P(1)' group which adopts a constrained conformation to fit this pocket, together with two water molecules interacting with the ST3-specific residue Gln215. These observations provide clues for the design of more specific inhibitors and show how ST3 can accommodate a phosphinic inhibitor mimicking a (d, l) peptide. The presence of a water molecule interacting with one oxygen atom of the inhibitor phosphinyl group and the proline residue of the Met-turn suggests how the intermediate formed during proteolysis may be stabilized. Furthermore, the hydrogen bond distance observed between the methyl of the phosphinic group and the carbonyl group of Ala182 mimics the interaction between this carbonyl group and the amide group of the cleaved peptidic bond. Our crystal structure provides a good model to study the MMPs mechanism of proteolysis.
Subject(s)
Metalloendopeptidases/chemistry , Oligopeptides/chemistry , Amino Acid Sequence , Binding Sites , Calcium/chemistry , Cations, Divalent/chemistry , Cations, Divalent/metabolism , Computer Simulation , Crystallography, X-Ray , Drug Design , Hydrogen Bonding , Ligands , Matrix Metalloproteinase 11 , Metalloendopeptidases/antagonists & inhibitors , Metalloendopeptidases/metabolism , Models, Molecular , Molecular Sequence Data , Oligopeptides/pharmacology , Protease Inhibitors/pharmacology , Sequence Alignment , Zinc/chemistryABSTRACT
Using the human basal transcription factors TFIID and TFIIH as examples, we show that pairwise coexpression of polypeptides in Escherichia coli can be used as a tool for the identification of specifically interacting subunits within multiprotein complexes. We find that coexpression of appropriate combinations generally leads to an increase in the solubility and stability of the polypeptides involved, which means that large amounts of the resulting complexes can immediately be obtained for subsequent biochemical and structural analysis. Furthermore, we demonstrate that the solubilization and/or the proper folding of a protein by its natural partner can be used as a monitor for deletion mapping to determine precise interaction domains. Coexpression can be used as an alternative or complementary approach to conventional techniques for interaction studies such as yeast two-hybrid analysis, GST pulldown and immunoprecipitation.
Subject(s)
Escherichia coli/genetics , Transcription Factors, TFII/chemistry , Transcription Factors, TFII/metabolism , Transcription Factors/chemistry , Transcription Factors/metabolism , Binding Sites , Gene Expression , Genetic Vectors/genetics , Humans , Macromolecular Substances , Models, Molecular , Protein Binding , Protein Folding , Protein Structure, Quaternary , Protein Subunits , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Deletion , Solubility , Transcription Factor TFIID , Transcription Factor TFIIH , Transcription Factors/genetics , Transcription Factors, TFII/genetics , Two-Hybrid System TechniquesABSTRACT
The crystal structure of a triple cysteine to serine mutant ERalpha ligand-binding domain (LBD), complexed with estradiol, shows that despite the presence of a tightly bound agonist ligand, the protein exhibits an antagonist-like conformation, similar to that observed in raloxifen and 4-hydroxytamoxifen-bound structures. This mutated receptor binds estradiol with wild type affinity and displays transcriptional activity upon estradiol stimulation, but with limited potency (about 50%). This partial activity is efficiently repressed in antagonist competition assays. The comparison with available LBD structures reveals key features governing the positioning of helix H12 and highlights the importance of cysteine residues in promoting an active conformation. Furthermore the present study reveals a hydrogen bond network connecting ligand binding to protein trans conformation. These observations support a dynamic view of H12 positioning, where the control of the equilibrium between two stable locations determines the partial agonist character of a given ligand.
Subject(s)
Receptors, Estrogen/chemistry , Receptors, Estrogen/metabolism , Cloning, Molecular , Crystallography, X-Ray , Estrogen Antagonists/pharmacology , Estrogen Receptor alpha , Humans , Hydrogen Bonding , Ligands , Models, Molecular , Mutagenesis, Site-Directed , Protein Conformation , Receptors, Estrogen/agonists , Receptors, Estrogen/geneticsABSTRACT
Vitamin D nuclear receptor mediates the genomic actions of the active form of vitamin D, 1,25(OH)2D3. This hormone is involved in calcium and phosphate metabolism and cell differentiation. Compared to other nuclear receptors, VDR presents a large insertion region at the N-terminal part of the ligand binding domain between helices H1 and H3, encoded by an additional exon. This region is poorly conserved in VDR in different species and is not well ordered as observed by secondary structure prediction. We engineered a VDR ligand binding domain mutant by removing this insertion region. Here we report its biochemical and biophysical characterization. The mutant protein exhibits the same ligand binding, dimerization with retinoid X receptor and transactivation properties as the wild-type VDR, suggesting that the insertion region does not affect these main functions. Solution studies by small angle X-ray scattering shows that the conformation in solution of the VDR mutant is similar to that observed in the crystal and that the insertion region in the VDR wild-type is not well ordered.
Subject(s)
Receptors, Calcitriol/genetics , Receptors, Calcitriol/physiology , Amino Acid Sequence , Animals , COS Cells , Calcitriol/analogs & derivatives , Calcitriol/metabolism , Humans , Ligands , Models, Molecular , Molecular Sequence Data , Protein Structure, Tertiary , Receptors, Calcitriol/chemistry , Sequence Deletion , Sequence Homology, Amino Acid , Transcriptional Activation , Transfection , X-Ray DiffractionABSTRACT
The presence of two short signature sequence motifs (His-Ile-Gly-His (HIGH) and Lys-Met-Ser-Lys (KMSK)) is a characteristic of the class I aminoacyl-tRNA synthetases. These motifs constitute a portion of the catalytic site in three dimensions and play an important role in catalysis. In particular, the second lysine of the KMSK motif (K2) is the crucial catalytic residue for stabilization of the transition state of the amino acid activation reaction (aminoacyl-adenylate formation). Arginyl-tRNA synthetase (ArgRS) is unique among all of the class I enyzmes, as the majority of ArgRS species lack canonical KMSK sequences. Thus, the mechanism by which this group of ArgRSs achieves the catalytic reaction is not well understood. Using three-dimensional modeling in combination with sequence analysis and site-directed mutagenesis, we found a conserved lysine in the KMSK-lacking ArgRSs upstream of the HIGH sequence motif, which is likely to be a functional counterpart of the canonical class I K2 lysine. The results suggest a plausible partition of the ArgRSs into two major groups, on the basis of the conservation of the HIGH lysine.
Subject(s)
Arginine-tRNA Ligase/metabolism , Lysine/metabolism , Amino Acid Sequence , Arginine-tRNA Ligase/chemistry , Catalysis , Models, Molecular , Molecular Sequence Data , Protein Conformation , Sequence Homology, Amino AcidABSTRACT
The major postembryonic developmental events happening in insect life, including molting and metamorphosis, are regulated and coordinated temporally by pulses of ecdysone. The biological activity of this steroid hormone is mediated by two nuclear receptors: the ecdysone receptor (EcR) and the Ultraspiracle protein (USP). The crystal structure of the ligand-binding domain from the lepidopteran Heliothis virescens USP reported here shows that the loop connecting helices H1 and H3 precludes the canonical agonist conformation. The key residues that stabilize this unique loop conformation are strictly conserved within the lepidopteran USP family. The presence of an unexpected bound ligand that drives an unusual antagonist conformation confirms the induced-fit mechanism accompanying the ligand binding. The ligand-binding pocket exhibits a retinoid X receptor-like anchoring part near a conserved arginine, which could interact with a USP ligand functional group. The structure of this receptor provides the template for designing inhibitors, which could be utilized as a novel type of environmentally safe insecticides.
Subject(s)
Insect Proteins/chemistry , Receptors, Retinoic Acid/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Animals , Crystallography, X-Ray , Insecta , Lepidoptera , Ligands , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Retinoid X Receptors , Sequence Homology, Amino AcidABSTRACT
Nuclear receptors are DNA-binding factors which regulate the transcription of sets of specific genes in response to cognate ligands, usually small lipophilic molecules, thus controlling numerous physiological events in development, procreation, homeostasis, and cellular life. Their ligand-dependent activity makes nuclear receptors obvious targets for drug design in many therapeutic areas. Crystallographic studies have revealed the structure of isolated domains but not, yet, of a whole protein, probably due to an intrinsic flexibility at work in nuclear receptor action. The structure of DNA-binding domain dimers in complex with an oligonucleotide has brought insights into how nuclear receptors recognize and bind to their target sequences ('response elements'). The structure of several ligand-binding domains in different ligation states has provided evidence for a ligand-dependent transcriptional switch and a molecular basis for the mode of action of agonists and antagonists.
Subject(s)
Receptors, Cytoplasmic and Nuclear/chemistry , Amino Acid Sequence , Animals , Binding Sites , Chromatin/metabolism , DNA/metabolism , Humans , Ligands , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Sequence Homology, Amino Acid , Transcription, GeneticABSTRACT
The 2.2 A crystal structure of a ternary complex formed by yeast arginyl-tRNA synthetase and its cognate tRNA(Arg) in the presence of the L-arginine substrate highlights new atomic features used for specific substrate recognition. This first example of an active complex formed by a class Ia aminoacyl-tRNA synthetase and its natural cognate tRNA illustrates additional strategies used for specific tRNA selection. The enzyme specifically recognizes the D-loop and the anticodon of the tRNA, and the mutually induced fit produces a conformation of the anticodon loop never seen before. Moreover, the anticodon binding triggers conformational changes in the catalytic center of the protein. The comparison with the 2.9 A structure of a binary complex formed by yeast arginyl-tRNA synthetase and tRNA(Arg) reveals that L-arginine binding controls the correct positioning of the CCA end of the tRNA(Arg). Important structural changes induced by substrate binding are observed in the enzyme. Several key residues of the active site play multiple roles in the catalytic pathway and thus highlight the structural dynamics of the aminoacylation reaction.