The ArcB Leucine Zipper Domain Is Required for Proper ArcB Signaling

The Arc two-component system modulates the expression of numerous genes in response to respiratory growth conditions. This system comprises ArcA as the response regulator and ArcB as the sensor kinase. ArcB is a tripartite histidine kinase whose activity is regulated by the oxidation of two cytosol-located redox-active cysteine residues that participate in intermolecular disulfide bond formation. Here, we report that the ArcB protein segment covering residues 70–121, fulfills the molecular characteristics of a leucine zipper containing coiled coil structure. Also, mutational analyses of this segment reveal three different phenotypical effects to be distributed along the coiled coil structure of ArcB, demonstrating that this motif is essential for proper ArcB signaling

Structural insights into the IgE mediated responses induced by the allergens Hev b 8 and Zea m 12 in their dimeric forms

"Oligomerization of allergens plays an important role in IgE-mediated reactions, as effective crosslinking of IgE-Fc epsilon RI complexes on the cell membrane is dependent on the number of exposed B-cell epitopes in a single allergen molecule or on the occurrence of identical epitopes in a symmetrical arrangement. Few studies have attempted to experimentally demonstrate the connection between allergen dimerization and the ability to trigger allergic reactions. Here we studied plant allergenic profilins rHev b 8 (rubber tree) and rZea m 12 (maize) because they represent an important example of cross-reactivity in the latex-pollen-food syndrome. Both allergens in their monomeric and dimeric states were isolated and characterized by exclusion chromatography and mass spectrometry and were used in immunological in vitro experiments. Their crystal structures were solved, and for Hev b 8 a disulfide-linked homodimer was found. Comparing the structures we established that the longest loop is relevant for recognition by IgE antibodies, whereas the conserved regions are important for cross-reactivity. We produced a novel monoclonal murine IgE (mAb 2F5), specific for rHev b 8, which was useful to provide evidence that profilin dimerization considerably increases the IgE-mediated degranulation in rat basophilic leukemia cells.

A Ribosomal Misincorporation of Lys for Arg in Human Triosephosphate Isomerase Expressed in Escherichia coli Gives Rise to Two Protein Populations

We previously observed that human homodimeric triosephosphate isomerase (HsTIM) expressed in Escherichia coli and purified to apparent homogeneity exhibits two significantly different thermal transitions. A detailed exploration of the phenomenon showed that the preparations contain two proteins; one has the expected theoretical mass, while the mass of the other is 28 Da lower. The two proteins were separated by size exclusion chromatography in 3 M urea. Both proteins correspond to HsTIM as shown by Tandem Mass Spectrometry (LC/ESI-MS/MS). The two proteins were present in nearly equimolar amounts under certain growth conditions. They were catalytically active, but differed in molecular mass, thermostability, susceptibility to urea and proteinase K. An analysis of the nucleotides in the human TIM gene revealed the presence of six codons that are not commonly used in E. coli. We examined if they were related to the formation of the two proteins. We found that expression of the enzyme in a strain that contains extra copies of genes that encode for tRNAs that frequently limit translation of heterologous proteins (Arg, Ile, Leu), as well as silent mutations of two consecutive rare Arg codons (positions 98 and 99), led to the exclusive production of the more stable protein. Further analysis by LC/ESI-MS/MS showed that the 28 Da mass difference is due to the substitution of a Lys for an Arg residue at position 99. Overall, our work shows that two proteins with different biochemical and biophysical properties that coexist in the same cell environment are translated from the same nucleotide sequence frame

Structural studies on threonyl and glycyl tRNA synthetases

L'analyse des différent états complexés de la ThrRS montre que la fixation de l'acide aminé déclenche un mouvement coordonné de la chaîne principale correspondant à 50 acides aminés. La fixation de l'ATP implique un mouvement important de la chaîne principale de l'autre côté du site catalytique et la rupture d'un pont salin. La boucle du motif 2, essentielle pour la première étape de la réaction, est également concernée. La fixation de l'ARNt requiert l'adaptation des régions déjà impliquées dans la fixation des petits substrats ainsi que d'une boucle qui vient stabiliser la conformation du complexe actif. La structure de la région N-terminale de la ThrRS de E. coli a permis de localiser le site de fixation de la sérine dans le site de correction des erreurs d'aminoacylation de l'ARNt spécifique de la thréonine par la sérine, mais son orientation exacte reste ambigüe, ce qui suggère le faible affinité du site. Dans la structure cristalline à 2.8 ? de résolution de la ThrRS de S.aureus, ce domaine contiendrait un ion métal. La ThrRS de E. coli réprime sa propre traduction en se fixant à un opérateur situé en amont du codon d'initiation. La structure du complexe entre le cœur catalytique de la ThrRS et le domaine essentiel de l'opérateur montre que le mode de reconnaissance de la boucle de l'anticodon de l'ARNt a été utilisé par le mRNA pour initier la fixation par l'enzyme. La position finale de l'opérateur repose sur un motif caractéristique du RNA, qui s'est adapté à la surface de l'enzyme pour générer un mécanisme de contrôle basé sur l'empêchement par gêne stérique de la fixation du ribosome. La structure de la sous-unité a de la GlyRS (tétramérique) d'E. coli montre que cette protéine consiste en une partie importante du cœur catalytique d'une aminoacyl-ARNt synthétase de classe II. Les motifs 1 et 2, qui n'étaient pas mis en évidence par l'alignement de séquences, y sont clairement présents. La structure se distingue de celle des autres enzymes de classe II par la présence de trois hélices situées au-dessus du feuillet ß antiparallèle canonique. La structure quaternaire de cette enzyme serait a2ß2, à la différence du cas (aß)2 représenté par la phénylalanyl-ARNt synthétase.The analysis of different complexed states on the system of ThrRS shows that the binding of the amino acid promotes a movement in the Ca position of 50 amino acids in one side of the catalytic domain. The binding of ATP triggers a movement in the Ca position of a salt bridge-locked region that is part of the core b-sheet cradling the small substrates. Two other small regions that surround the catalytic site move due to the small substrate binding in a cooperative way. The tRNA interacts with all four of these loops and several residues initially bound to threonine or ATP switch to a position in which they can contact the tRNA. The crystal structures of S. aureus ThrRS and of the N-terminal region of E. coli ThrRS show the presence of one metal ion and of a putative serine in the editing site, respectively. The role of the metal ion and the exact orientation of the amino acid could not be determined unambiguously, suggesting the low affinity of the sites.E. coli ThrRS represses its own translation by binding to an operator located upstream of the initiation codon. The crystal structure of the complex between the core of ThrRS and the essential domain of the operator shows that the recognition mode of the tRNA anticodon loop has been utilized by the mRNA to initiate the binding. The final position of the operator, upon which the control mechanism is based, relies on a characteristic RNA motif adapted to the enzyme surface.The crystal structure of the a-subunit of E. coli GlyRS shows that this protein forms most of the canonic catalytic core of the class II tRNA synthetases. This subunit shows unambiguously the presence of motif 1 and 2, difficult to infer at the sequence level. The structure differs from the core of other class II aaRS by the presence of three helices covering 80 residues of the C-terminal region and situated on top of the antiparallel b strand. The quaternary structure of this enzyme would be a2b2, in contrast with the (ab)2 case presented in PheRS.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Conformational movements and cooperativity upon amino acid, ATP and tRNA binding in threonyl-tRNA synthetase

The crystal structures of threonyl-tRNA synthetase (ThrRS) from Staphylococcus aureus, with ATP and an analogue of threonyl adenylate, are described. Together with the previously determined structures of Escherichia coli ThrRS with different substrates, they allow a comprehensive analysis of the effect of binding of all the substrates: threonine, ATP and tRNA. The tRNA, by inserting its acceptor arm between the N-terminal domain and the catalytic domain, causes a large rotation of the former. Within the catalytic domain, four regions surrounding the active site display significant conformational changes upon binding of the different substrates. The binding of threonine induces the movement of as much as 50 consecutive amino acid residues. The binding of ATP triggers a displacement, as large as 8 Å at some C<SUP>α</SUP> positions, of a strand-loop-strand region of the core β-sheet. Two other regions move in a cooperative way upon binding of threonine or ATP: the motif 2 loop, which plays an essential role in the first step of the aminoacylation reaction, and the ordering loop, which closes on the active site cavity when the substrates are in place. The tRNA interacts with all four mobile regions, several residues initially bound to threonine or ATP switching to a position in which they can contact the tRNA. Three such conformational switches could be identified, each of them in a different mobile region. The structural analysis suggests that, while the small substrates can bind in any order, they must be in place before productive tRNA binding can occur

Achieving Error-Free Translation: The Mechanism of Proofreading of Threonyl-tRNA Synthetase at Atomic Resolution

The fidelity of aminoacylation of tRNAThr by the threonyl-tRNA synthetase (ThrRS) requires the discrimination of the cognate substrate threonine from the noncognate serine. Misacylation by serine is corrected in a proofreading or editing step. An editing site has been located 39 Å away from the aminoacylation site. We report the crystal structures of this editing domain in its apo form and in complex with the serine product, and with two nonhydrolyzable analogs of potential substrates: the terminal tRNA adenosine charged with serine, and seryl adenylate. The structures show how serine is recognized, and threonine rejected, and provide the structural basis for the editing mechanism, a water-mediated hydrolysis of the mischarged tRNA. When the adenylate analog binds in the editing site, a phosphate oxygen takes the place of one of the catalytic water molecules, thereby blocking the reaction. This rules out a correction mechanism that would occur before the binding of the amino acid on the tRNA

Identification of amino acids that account for long-range interactions in two triosephosphate isomerases from pathogenic trypanosomes.

For a better comprehension of the structure-function relationship in proteins it is necessary to identify the amino acids that are relevant for measurable protein functions. Because of the numerous contacts that amino acids establish within proteins and the cooperative nature of their interactions, it is difficult to achieve this goal. Thus, the study of protein-ligand interactions is usually focused on local environmental structural differences. Here, using a pair of triosephosphate isomerase enzymes with extremely high homology from two different organisms, we demonstrate that the control of a seventy-fold difference in reactivity of the interface cysteine is located in several amino acids from two structurally unrelated regions that do not contact the cysteine sensitive to the sulfhydryl reagent methylmethane sulfonate, nor the residues in its immediate vicinity. The change in reactivity is due to an increase in the apparent pKa of the interface cysteine produced by the mutated residues. Our work, which involved grafting systematically portions of one protein into the other protein, revealed unsuspected and multisite long-range interactions that modulate the properties of the interface cysteines and has general implications for future studies on protein structure-function relationships

Estudio de las interacciones entre las subunidades ASA del brazo periférico de la ATP sintasa mitocondrial de Polytomella sp

La ATP sintasa mitocondrial de las algas clorofíceas ha perdido una serie de subunidades clásicas que están involucradas en la formación del cuello lateral (estator) de la enzima y en la dimerización de la misma. En compensación, ha adquirido 9 subunidades novedosas, de origen evolutivo desconocido, que han sido llamadas ASA1 a ASA9. Estas subunidades ASA solamente están presentes en el grupo de las algas clorofíceas y no se encuentran en otras algas cercanamente relacionadas, como las algas verdes del linaje de las ulvofíceas, de las prasinofíceas o de las trebuxiofíceas. Experimentos de disociación de la enzima, tratamiento con agentes entrecruzadores y estudios estructurales llevaron a la propuesta de un modelo estructural de esta ATP sintasa, donde se propone que las subunidades ASA1 a 9 participan en la estructura del estator periférico y en la dimerización de la misma. En el presente trabajo se desea abordar un estudio más detallado de las subunidades ASA, para conocer acerca de las interacciones que establecen entre ellas y como contribuyen a la formación del brazo periférico de la enzima. La estrategia experimental parte de la clonación de los genes de las subunidades en vectores de expresión en bacteria, la expresión de las subunidades recombinantes y su purificación. Los estudios de inmunoréplica indican las interacciones entre las subunidades ASA4-ASA7 y ASA4-ASA2

Selection of a Single Domain Antibody, Specific for an HLA-Bound Epitope of the Mycobacterial Ag85B Antigen

T cells recognizing epitopes on the surface of mycobacteria-infected macrophages can impart protection, but with associated risk for reactivation to lung pathology. We aimed to identify antibodies specific to such epitopes, which carry potentials for development toward novel therapeutic constructs. Since epitopes presented in the context of major histocompatibility complex alleles are rarely recognized by naturally produced antibodies, we used a phage display library for the identification of monoclonal human single domain antibody producing clones. The selected 2C clone displayed T cell receptor-like recognition of an HLA-A*0201 bound(199)KLVANNTRL(207)peptide from the Ag85B antigen, which is known to be an immunodominant epitope for human T cells. The specificity of the selected domain antibody was demonstrated by solid phase immunoassay and by immunofluorescent surface staining of peptide loaded cells of the T2 cell line. The antibody affinity binding was determined by biolayer interferometry. Our results validated the used technologies as suitable for the generation of antibodies against epitopes on the surface ofMycobacterium tuberculosisinfected cells. The potential approaches forward the development of antibody in immunotherapy of tuberculosis have been outlined in the discussion.Immunogenetics and cellular immunology of bacterial infectious disease