Recognition of l--homomethionine by methionyl-trna synthetase

Living cells commonly employ the 20 canonical amino acids for ribosomal protein synthesis, although a few other amino acids (such as selenocysteine or pyrrolysine) are also employed in rare cases. The natural repertoire of amino acids is therefore rather narrow. This limits the possibilities to engineer non-natural proteins with new properties, usable for therapeutic or industrial purpose. Therefore, a dominant trend in the field of protein engineering is to build up systems for incorporating non-natural amino acids during protein biosynthesis in vivo (reviewed in Xiao and Schultz, 2016). Although this strategy has been successful in several cases, all amino acids used to date were amino acids. Site-specific incorporation of a amino acid in a polypeptide would introduce unprecedented flexibility in the main chain, thereby enlarging the geometric possibilities for protein folding. It has recently been demonstrated, using an in vitro cell-free translation system, that the ribosome is able to efficiently incorporate amino acids at discrete sites in a polypeptide (Fujino et al, 2016). At this stage, a technological issue that needs to be resolved for in vivo synthesis of a protein containing amino acid residues is the design of aminoacyl-tRNA synthetases able to efficiently esterify the desired amino acids onto specific transfer RNAs. A recent study has shown that some E. coli aminoacyl-tRNA synthetases are indeed able to use amino acids as substrates (Melo Czekster et al, 2016). Our final goal is to optimize incorporation of methionine in polypeptides. To this aim, we have first characterized the binding and activation of L--homomethionine by E. coli methionyl-tRNA synthetase. It is demonstrated that activation of the amino acid occurs but at a markedly lower rate than activation of L--methionine. A 1.45 Å crystal structure of methionyl-tRNA synthetase complexed with the amino acid will also be presented. We will discuss how this structure is used for optimizing aminoacylation of tRNA with L--homomethionine. Fujino, T., Goto, Y., Suga, H. and Murakami, H. J Am Chem Soc, 2016, 138, 1962-1969. Melo Czekster, C., Robertson, W. E., Walker, A. S., Soll, D. and Schepartz, A. J Am Chem Soc, 2016, 138, 5194-5197. Cold Spring Harb Perspect Biol, 2016, vol8-9

Discovery of Escherichia coli methionyl-tRNA synthetase mutants for efficient labeling of proteins with azidonorleucine in vivo

Incorporation of noncanonical amino acids into cellular proteins often requires engineering new aminoacyl-tRNA synthetase activity into the cell. A screening strategy that relies on cell-surface display of reactive amino acid side-chains was used to identify a diverse set of methionyl-tRNA synthetase (MetRS) mutants that allow efficient incorporation of the methionine (Met) analog azidonorleucine (Anl). We demonstrate that the extent of cell-surface labeling in vivo is a good indicator of the rate of Anl activation by the MetRS variant harbored by the cell. By screening at low Anl concentrations in Met-supplemented media, MetRS variants with improved activities toward Anl and better discrimination against Met were identified

Boosting endoplasmic reticulum folding capacity reduces unfolded protein response activation and intracellular accumulation of human kidney anion exchanger 1 in Saccharomyces cerevisiae

Human kidney anion exchanger 1 (kAE1) facilitates simultaneous efflux of bicarbonate and absorption of chloride at the basolateral membrane of α-intercalated cells. In these cells, kAE1 contributes to systemic acid–base balance along with the proton pump v-H+-ATPase and the cytosolic carbonic anhydrase II. Recent electron microscopy analyses in yeast demonstrate that heterologous expression of several kAE1 variants causes a massive accumulation of the anion transporter in intracellular membrane structures. Here, we examined the origin of these kAE1 aggregations in more detail. Using various biochemical techniques and advanced light and electron microscopy, we showed that accumulation of kAE1 mainly occurs in endoplasmic reticulum (ER) membranes which eventually leads to strong unfolded protein response (UPR) activation and severe growth defect in kAE1 expressing yeast cells. Furthermore, our data indicate that UPR activation is dose dependent and uncoupled from the bicarbonate transport activity. By using truncated kAE1 variants, we identified the C-terminal region of kAE1 as crucial factor for the increased ER stress level. Finally, a redistribution of ER-localized kAE1 to the cell periphery was achieved by boosting the ER folding capacity. Our findings not only demonstrate a promising strategy for preventing intracellular kAE1 accumulation and improving kAE1 plasma membrane targeting but also highlight the versatility of yeast as model to investigate kAE1-related research questions including the analysis of structural features, protein degradation and trafficking. Furthermore, our approach might be a promising strategy for future analyses to further optimize the cell surface targeting of other disease-related PM proteins, not only in yeast but also in mammalian cells. Take Away We analysed the intracellular transport of human kAE1 to the yeast plasma membrane. We studied the effect of human kAE1 expression on yeast growth and UPR activation. We investigated the impact of different kAE1 truncation variants on UPR induction We implemented intervention strategies to improve PM targeting of kAE1

Identification of a second GTP-bound magnesium ion in archaeal initiation factor 2

International audienceEukaryotic and archaeal translation initiation processes involve a heterotrimeric GTPase e/aIF2 crucial for accuracy of start codon selection. In eu-karyotes, the GTPase activity of eIF2 is assisted by a GTPase-activating protein (GAP), eIF5. In ar-chaea, orthologs of eIF5 are not found and aIF2 GT-Pase activity is thought to be non-assisted. However , no in vitro GTPase activity of the archaeal factor has been reported to date. Here, we show that aIF2 significantly hydrolyses GTP in vitro. Within aIF2␥, H97, corresponding to the catalytic histidine found in other translational GTPases, and D19, from the GKT loop, both participate in this activity. Several high-resolution crystal structures were determined to get insight into GTP hydrolysis by aIF2␥. In particular, a crystal structure of the H97A mutant was obtained in the presence of non-hydrolyzed GTP. This structure reveals the presence of a second magnesium ion bound to GTP and D19. Quantum chemical/molecular mechanical simulations support the idea that the second magnesium ion may assist GTP hydrolysis by helping to neutralize the developing negative charge in the transition state. These results are discussed in light of the absence of an identified GAP in archaea to assist GTP hydrolysis on aIF2

Roles of yeast eIF2α and eIF2β subunits in the binding of the initiator methionyl-tRNA

International audienceHeterotrimeric eukaryotic/archaeal translation initiation factor 2 (e/aIF2) binds initiator methionyl-tRNA and plays a key role in the selection of the start codon on messenger RNA. tRNA binding was extensively studied in the archaeal system. The γ subunit is able to bind tRNA, but the α subunit is required to reach high affinity whereas the β subunit has only a minor role. In Saccharomyces cerevisiae however, the available data suggest an opposite scenario with β having the most important contribution to tRNA-binding affinity. In order to overcome difficulties with purification of the yeast eIF2γ subunit, we designed chimeric eIF2 by assembling yeast α and β subunits to archaeal γ subunit. We show that the β subunit of yeast has indeed an important role, with the eukaryote-specific N- and C-terminal domains being necessary to obtain full tRNA-binding affinity. The α subunit apparently has a modest contribution. However, the positive effect of α on tRNA binding can be progressively increased upon shortening the acidic C-terminal extension. These results, together with small angle X-ray scattering experiments, support the idea that in yeast eIF2, the tRNA molecule is bound by the α subunit in a manner similar to that observed in the archaeal aIF2-GDPNP-tRNA complex. © The Author(s) 2012. Published by Oxford University Press

La motte castrale de Guéramé à Courgains (Sarthe), aux confins du Maine et du Perche

Une opération de diagnostic réalisée en 2006 sur la commune de Courgains dans le nord de la Sarthe a permis de confirmer la présence d’une motte et de sa basse-cour, site découvert en prospection aérienne en 1999 par G. Leroux (INRAP). Le gisement, baptisé « Guéramé » par son inventeur, est d’abord constitué d’un fossé de 7 à 8 m de largeur et de presque 2,50 m de profondeur. Il entoure une plate-forme circulaire de 20 m de diamètre. Aujourd’hui, le relief de la motte ainsi enserrée est presq..

Cdc123, a Cell Cycle Regulator Needed for eIF2 Assembly, Is an ATP-Grasp Protein with Unique Features

International audienceEukaryotic initiation factor 2 (eIF2), a heterotrimeric guanosine triphosphatase, has a central role in protein biosynthesis by supplying methionylated initiator tRNA to the ribosomal translation initiation complex and by serving as a target for translational control in response to stress. Recent work identified a novel step indispensable for eIF2 function: assembly of eIF2 from its three subunits by the cell proliferation protein Cdc123. We report the first crystal structure of a Cdc123 representative, that from Schizosaccharomyces pombe, both isolated and bound to domain III of Saccharomyces cerevisiae eIF2 gamma. The structures show that Cdc123 resembles enzymes of the ATP-grasp family. Indeed, Cdc123 binds ATP-Mg2+, and conserved residues contacting ATP-Mg2+ are essential for Cdc123 to support eIF2 assembly and cell viability. A docking of eIF2 alpha gamma onto Cdc123, combined with genetic and biochemical experiments, allows us to propose a model explaining how Cdc123 participates in the biogenesis of eIF2 through facilitating assembly of eIF2 gamma to eIF2 alpha

Switching from an Induced-Fit to a Lock-and-Key Mechanism in an Aminoacyl-tRNA Synthetase with Modified Specificity

Methionyl-tRNA synthetase (MetRS) specifically binds its methionine substrate in an induced-fit mechanism, with methionine binding causing large rearrangements. Mutated MetRS able to efficiently aminoacylate the methionine (Met) analog azidonorleucine (Anl) have been identified by saturation mutagenesis combined with in vivo screening procedures. Here, the crystal structure of such a mutated MetRS was determined in the apo form as well as complexed with Met or Anl (1.4 to 1.7 Å resolution) to reveal the structural basis for the altered specificity. The mutations result in both the loss of important contacts with Met and the creation of new contacts with Anl, thereby explaining the specificity shift. Surprisingly, the conformation induced by Met binding in wild-type MetRS already occurs in the apo form of the mutant enzyme. Therefore, the mutations cause the enzyme to switch from an induced-fit mechanism to a lock-and-key one, thereby enhancing its catalytic efficiency

Unravelling the mechanism of non-ribosomal peptide synthesis by cyclodipeptide synthases.

International audienceCyclodipeptide synthases form cyclodipeptides from two aminoacyl transfer RNAs. They use a ping-pong mechanism that begins with transfer of the aminoacyl moiety of the first aminoacyl tRNA onto a conserved serine, yielding an aminoacyl enzyme. Combining X-ray crystallography, site-directed mutagenesis and affinity labelling of the cyclodipeptide synthase AlbC, we demonstrate that the covalent intermediate reacts with the aminoacyl moiety of the second aminoacyl tRNA, forming a dipeptidyl enzyme, and identify the aminoacyl-binding sites of the aminoacyl tRNAs

Fluctuations in Parkinson’s disease and personalized medicine: bridging the gap with the neuropsychiatric fluctuation scale

BackgroundNeuropsychiatric fluctuations (NpsyF) are frequent and disabling in people with Parkinson’s disease (PD). In OFF-medication, NpsyF entail minus neuropsychiatric symptoms (NPS) like anxiety, apathy, sadness, and fatigue. In ON-medication, NpsyF consist in plus NPS, such as high mood, hypomania, and hyperactivity. Accurate identification of these NpsyF is essential to optimize the overall PD management. Due to lack of punctual scales, the neuropsychiatric fluctuation scale (NFS) has been recently designed to assess NpsyF in real time. The NFS comprises 20 items with two subscores for plus and minus NPS, and a total score.ObjectiveTo evaluate the psychometric properties of the NFS in PD.MethodsPD patients with motor fluctuations and healthy controls (HC) were assessed. In PD patients, the NFS was administrated in both the ON-and OFF-medication conditions, together with the movement disorders society-unified Parkinson disease rating scale parts I–IV. Depression (Beck depression scale II), apathy (Starkstein apathy scale) and non-motor fluctuations items of the Ardouin scale of behaviour in PD (ASBPD OFF and ON items) were also assessed. NFS internal structure was evaluated with principal component analysis consistency (PCA) in both medication conditions in PD patients and before emotional induction in HC. NFS internal consistency was assessed using Cronbach’s alpha coefficient. NFS convergent and divergent validity was measured through correlations with BDI-II, Starktein, and ASBPD OFF and ON non motor items. Specificity was assessed comparing NFS global score between the HC and PD populations. Sensitivity was evaluated with t-student test comparing the ON-and the OFF-medication conditions for NFS global score and for minus and plus subscores.ResultsIn total, 101 consecutive PD patients and 181 HC were included. In PD patients and HC, PCA highlighted one component that explained 32–35 and 42% of the variance, respectively. Internal consistency was good for both the NFS-plus (alpha =0.88) and NFS-minus items (alpha =0.8). The NFS showed a good specifity for PD (p < 0.0001) and a good sensitivity to the medication condition (p < 0.0001).ConclusionThe satisfactory properties of the NFS support its use to assess acute neuropsychiatric fluctuations in PD patients, adding to available tools