Catalytic mechanism of the tryptophan activation reaction revealed by crystal structures of human tryptophanyl-tRNA synthetase in different enzymatic states

Human tryptophanyl-tRNA synthetase (hTrpRS) differs from its bacterial counterpart at several key positions of the catalytic active site and has an extra N-terminal domain, implying possibly a different catalytic mechanism. We report here the crystal structures of hTrpRS in complexes with Trp, tryptophanamide and ATP and tryptophanyl-AMP, respectively, which represent three different enzymatic states of the Trp activation reaction. Analyses of these structures reveal the molecular basis of the mechanisms of the substrate recognition and the activation reaction. The dimeric hTrpRS is structurally and functionally asymmetric with half-of-the-sites reactivity. Recognition of Trp is by an induced-fit mechanism involving conformational change of the AIDQ motif that creates a perfect pocket for the binding and activation of Trp and causes coupled movements of the N-terminal and C-terminal domains. The KMSAS loop appears to have an inherent flexibility and the binding of ATP stabilizes it in a closed conformation that secures the position of ATP for catalysis. Our structural data indicate that the catalytic mechanism of the Trp activation reaction by hTrpRS involves more moderate conformational changes of the structural elements at the active site to recognize and bind the substrates, which is more complex and fine-tuned than that of bacterial TrpRS

Functional Expression of Human Adenine Nucleotide Translocase 4 in Saccharomyces Cerevisiae

The adenine nucleotide translocase (ANT) mediates the exchange of ADP and ATP across the inner mitochondrial membrane. The human genome encodes multiple ANT isoforms that are expressed in a tissue-specific manner. Recently a novel germ cell-specific member of the ANT family, ANT4 (SLC25A31) was identified. Although it is known that targeted depletion of ANT4 in mice resulted in male infertility, the functional biochemical differences between ANT4 and other somatic ANT isoforms remain undetermined. To gain insight into ANT4, we expressed human ANT4 (hANT4) in yeast mitochondria. Unlike the somatic ANT proteins, expression of hANT4 failed to complement an AAC-deficient yeast strain for growth on media requiring mitochondrial respiration. Moreover, overexpression of hANT4 from a multi-copy plasmid interfered with optimal yeast growth. However, mutation of specific amino acids of hANT4 improved yeast mitochondrial expression and supported growth of the AAC-deficient yeast on non-fermentable carbon sources. The mutations affected amino acids predicted to interact with phospholipids, suggesting the importance of lipid interactions for function of this protein. Each mutant hANT4 and the somatic hANTs exhibited similar ADP/ATP exchange kinetics. These data define common and distinct biochemical characteristics of ANT4 in comparison to ANT1, 2 and 3 providing a basis for study of its unique adaptation to germ cells

Etudes cinetiques de la reaction d'activation du tryptophane catalysee par la tryptophanyl-tARN synthetase de Escherichia coli et d'aminoacylation du tARNTrp de boeuf catalysee par la tryptophanyl-tARN synthetase de boeuf

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Le transporteur mitochondrial de nucléotides adényliques de la levure S. cerevisiae (contribution de chaque sous-unité à l'organisation membranaire et à la fonction de transport)

Le transporteur mitochondrial de nucléotides adényliques (Ancp) est une protéine de 32 kDa localisée dans la membrane interne. Elle appartient à la famille des transporteurs mitochondriaux. Elle permet l'échange de l'ADP contre de l'ATP entre le cytoplasme et la matrice. L'Ancp possède 6 segments transmembranaires potentiellement organisés en hélices a reliées par des boucles hydrophiles. De nombreuses études ont montré, que, in vivo, l'Ancp se trouve sous forme dimérique. Ce travail a consisté à étudier le rôle de chacune de ces sous-unités dans la fonction de transport et dans la structure de l'unité fonctionnelle. Pour cela, nous avons constrtuit des gènes permettant l'expression de deux types d'hétérodimères en tandem. Dans ces chimères, un des protomères est de type sauvage (Anc2) et l'autre est soit un mutant inactif de ANC2 (op1) soit un autre membre de la famille des transporteurs mitochondriaux : le transporteur de phosphate (PiC). Les constructions ANC2/op1 et PiC/ANC2 ont été respectivement introduites dans une souche delta anc2 et une souche delta pic, delta anc2. Ces gènes restaurent la croissance sur lactate de façon comparable à ANC2 sauvage. De plus, Anc2-Picp et Pic-Anc2p restaurent de façon simultanée l'échange ADP/ATP et le transport du Pi. L'expoession in vivo de ces gènes conduit à des protéines de 64 kDa. Les paramètres cinétiques de l'échange ADP/ATP ainsi que ceux de la liaison d'un inhibiteur spécifique de l'Ancp, l'atractylate, ont été comparés à ceux de Anc2p. Nos résultats expérimentaux indiquent que les monomères coopèrent lors de l'échange des nucléotides. De plus, les propriétés de la chimère de PiC permettent de conclure quant à la structure tertiaire du monomère dans la membrane mitochondriale.The adenine nucleotide carrier (Ancp) is a 32 kDa mitochondrial protein located in the inner membrane. It is a member of the mitochondrial carrier family. Ancp allows the exchange of ADP versus ATP between cytoplasm and matrix. It contains six transmembrane segments organized in alpha helix and connected via hydrophilic loops. Numerous studies have shown the endogenous Ancp to be a dimer in vivo. To study the structural folding and functional properties of each monomer within the diner, we have constructed genes encoding two types of covalent tandem heterodimers. In these chimeras, one of the subunits is of the wild type and the other is either an inactive mutant of ANC2 (op1) or a member of the mitochondrial carrier family, namely the phosphate carrier (PiC). The ANC2/op1 and the PiC/ANC2 contructs were respectively introduced into a delta anc2 and a double deleted delta pic, delta anc2 mutant strains and then the restoration of growth on non-fermentable carbon sources was studied. Results indicate that Anc2-op1p and op1-Anc2p were as efficient as Anc2p. Moreover, Anc2-Picp and Pic-Anc2p were able to simultaneously restore both the ADP/ATP and Pi transport functions. In vivo, expression of the dimeric genes led to synthesis of stable 64 kDa proteins. Binding properties of [3H] ATR, a specific inhibitor of Ancp, and ADP/ATP exchange kinetics were compared with those of Anc2p. Our results indicate that protomers of the op1/Anc2p chimera cross talk to each other during the nucleotide exchange. Furthermore, the properties of PiC chimera have allowed us to conclude about the tertiary structure of the monomer in the mitochondrial inner membrane.BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF

Le transporteur ADP/ATP mitochondrial (études fonctionnelles des prolines des hélices transmembranaires 1, 3 et 5 et étude des conformations associées au transport de nucléotides)

Le transporteur mitochondrial de nucléotides adényliques (Ancp), localisé dans la membrane interne mitochondriale, catalyse l'échange ADP/ATP entre le cytoplasme et la matrice mitochondriale. Il lie deux classes d'inhibiteurs naturels avec une grande spécificité et une haute affinité. Ces deux types d'inhibiteurs, BA et CATR, stabilisent Ancp dans deux conformations distinctes impliquées dans le transport des nucléotides. La compréhension des changements conformationnels subits par Ancp est essentielle pour décrire précisément le mécanisme d'échange des nucléotides. La structure atomique du transporteur de bœuf a montré que les hélices transmembranaires 1, 3 et 5 sont coudées par la présence de prolines qui pourraient donc être impliquées dans les changements conformationnels associés au transport.Dans la première partie de ce manuscrit, ces prolines ont été mutées en alanine ou en leucine et les conséquences de ces mutations ont été étudiées au niveau de la cellule (phénotype, morphologie, contenu en protéines) et des mitochondries en examinant le transport lui-même ainsi que toutes les fonctions mitochondriales (respiration, contenu en protéines, importation des protéines, morphologie ). Il peut-être conclu de ces études que ces prolines jouent un rôle dans le transport mais également dans la biogenèse mitochondriale (import d'Ancp, équilibre fusion/fission mitochondriale).Dans la deuxième partie, ont été étudiées des mutations qui stabilisent Ancp dans la conformation BA ou CATR. L'objectif était d'obtenir des formes stables du transporteur représentant des états intermédiaires du transport pour en étudier la structure atomique par cristallographie. Les résultats préliminaires sont prometteurs.The mitochondrial ADP/ATP carrier (Ancp), located in the inner mitochondrial membrane, catalyzes the ADP/ATP exchange between the cytoplasm and the mitochondrial membrane. Two classes of natural inhibitors can bind to the carrier with high specificity and affinity. These two families of inhibitors, BA and CATR, stabilize Ancp in two different conformations, which are involved in the nucleotide transport. Understanding the conformational changes undergone by Ancp is essential to describe precisely the nucleotide exchange mechanism. The atomic structure of the Beef Ancp unveiled kinks in transmembrane helices 1, 3 and 5 induced by the prolines, which therefore could be involved in the conformational changes associated with the nucleotide transport. In the first part of this manuscript, the three prolines were mutated into alanine or leucine and the results of these mutations were studied at the level of the cell (phenotype, morphology, protein content) and of the mitochondria by examining the transport itself and various mitochondria functions (respiration, protein content, protein import, morphology...). It can be concluded from these studies that these prolines play a key role in the transport but also in mitochondria biogenesis (Ancp import, mitochondrial fusion/fission balance).In the second part were studied mutations that stabilize Ancp in BA or CATR conformation. The goal was to obtain stable forms of Ancp that would correspond to intermediate steps of the transport to study their atomic structure by crystallography. The preliminary results are promising.BORDEAUX2-Bib. électronique (335229905) / SudocSudocFranceF

On the mechanism of tRNATrp aminoacylation catalysed by beef tryptophanyl-tRNA synthetase using presteady-state kinetics

AbstractThe dimeric tryptophanyl-tRNA synthetase from beef pancreas has been found to activate 2 tryptophans/mol enzyme [Eur. J. Biochem. (1982) 128, 389–398]. By using quenched-flow and stopped-flow methods under presteady-state conditions, we show that only one enzyme subunit operates at a time in the aminoacylation of tRNATrp and that the transfer reaction is not the rate-limiting step in the overall aminoacylation process

Etude des possibilites de realisation d'un micromoteur a electrets

Action concertee: MecaniqueSIGLEAvailable from Centre de Documentation Scientifique et Technique, CNRS, 26 rue Boyer, 75971 Paris Cedex 20 (France) / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc