Reproducibility and accuracy of microscale thermophoresis in the NanoTemper Monolith: a multi laboratory benchmark study.

Microscale thermophoresis (MST), and the closely related Temperature Related Intensity Change (TRIC), are synonyms for a recently developed measurement technique in the field of biophysics to quantify biomolecular interactions, using the (capillary-based) NanoTemper Monolith and (multiwell plate-based) Dianthus instruments. Although this technique has been extensively used within the scientific community due to its low sample consumption, ease of use, and ubiquitous applicability, MST/TRIC has not enjoyed the unambiguous acceptance from biophysicists afforded to other biophysical techniques like isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR). This might be attributed to several facts, e.g., that various (not fully understood) effects are contributing to the signal, that the technique is licensed to only a single instrument developer, NanoTemper Technology, and that its reliability and reproducibility have never been tested independently and systematically. Thus, a working group of ARBRE-MOBIEU has set up a benchmark study on MST/TRIC to assess this technique as a method to characterize biomolecular interactions. Here we present the results of this study involving 32 scientific groups within Europe and two groups from the US, carrying out experiments on 40 Monolith instruments, employing a standard operation procedure and centrally prepared samples. A protein-small molecule interaction, a newly developed protein-protein interaction system and a pure dye were used as test systems. We characterized the instrument properties and evaluated instrument performance, reproducibility, the effect of different analysis tools, the influence of the experimenter during data analysis, and thus the overall reliability of this method.

Interplay between mistranslation and oxidative stress in Escherichia coli.

Mistakes in translation are mostly associated with toxic effects in the cell due to the production of functionally aberrant and misfolded proteins. However, under certain circumstances mistranslation can have beneficial effects and enable cells to preadapt to other stress conditions. Mistranslation may be caused by mistakes made by aminoacyl-tRNA synthetases, essential enzymes that link amino acids to cognate tRNAs. There is an Escherichia coli strain expressing isoleucyl-tRNA synthetase mutant variant with inactivated editing domain which produces mistranslated proteomes where valine (Val) and norvaline (Nva) are misincorporated into proteins instead of isoleucine. We compared this strain with the wild-type to determine the effects of such mistranslation on bacterial growth in oxidative stress conditions. When the cells were pre-incubated with 0.75 mmol/L Nva or 1.5 mmol/L Val or Nva and exposed to hydrogen peroxide, no beneficial effect of mistranslation was observed. However, when the editing-deficient strain was cultivated in medium supplemented with 0.75 mmol/L Val up to the early or mid-exponential phase of growth and then exposed to oxidative stress, it slightly outgrew the wild-type grown in the same conditions. Our results therefore show a modest adaptive effect of isoleucine mistranslation on bacterial growth in oxidative stress, but only in specific conditions. This points to a delicate balance between deleterious and beneficial effects of mistranslation.

Protein-Protein Interactions of Seryl-tRNA Synthetases with Emphasis on Human Counterparts and Their Connection to Health and Disease.

Seryl-tRNA synthetases (SerRSs), members of the aminoacyl-tRNA synthetase family, interact with diverse proteins, enabling SerRSs to enhance their role in the translation of the genetic message or to perform alternative functions in cellular processes beyond translation. Atypical archaeal SerRS interacts with arginyl-tRNA synthetase and proteins of the ribosomal P-stalk to optimize translation through tRNA channeling. The complex between yeast SerRS and peroxin Pex21p provides a connection between translation and peroxisome function. The partnership between Arabidopsis SerRS and BEN1 indicates a link between translation and brassinosteroid metabolism and may be relevant in plant stress response mechanisms. In Drosophila, the unusual heterodimeric mitochondrial SerRS coordinates mitochondrial translation and replication via interaction with LON protease. Evolutionarily conserved interactions of yeast and human SerRSs with m3C32 tRNA methyltransferases indicate coordination between tRNA modification and aminoacylation in the cytosol and mitochondria. Human cytosolic SerRS is a cellular hub protein connecting translation to vascular development, angiogenesis, lipogenesis, and telomere maintenance. When translocated to the nucleus, SerRS acts as a master negative regulator of VEGFA gene expression. SerRS alone or in complex with YY1 and SIRT2 competes with activating transcription factors NFκB1 and c-Myc, resulting in balanced VEGFA expression important for proper vascular development and angiogenesis. In hypoxia, SerRS phosphorylation diminishes its binding to the VEGFA promoter, while the lack of nutrients triggers SerRS glycosylation, reducing its nuclear localization. Additionally, SerRS binds telomeric DNA and cooperates with the shelterin protein POT1 to regulate telomere length and cellular senescence. As an antitumor and antiangiogenic factor, human cytosolic SerRS appears to be a promising drug target and therapeutic agent for treating cancer, cardiovascular diseases, and possibly obesity and aging.

Substrate recognition and fidelity of maize seryl-tRNA synthetases.

Aminoacyl-tRNA synthetases (aaRSs) catalyze the attachment of amino acids to their cognate tRNAs to establish the genetic code. To obtain the high degree of accuracy that is observed in translation, these enzymes must exhibit strict substrate specificity for their cognate amino acids and tRNAs. We studied the requirements for tRNA(Ser) recognition by maize cytosolic seryl-tRNA synthetase (SerRS). The enzyme efficiently recognized bacterial and eukaryotic tRNAs(Ser) indicating that it can accommodate various types of tRNA(Ser) structures. Discriminator base G73 is crucial for recognition by cytosolic SerRS. Although cytosolic SerRS efficiently recognized bacterial tRNAs(Ser), it is localized exclusively in the cytosol. The fidelity of maize cytosolic and dually targeted organellar SerRS with respect to amino acid recognition was compared. Organellar SerRS exhibited higher discrimination against tested non-cognate substrates as compared with cytosolic counterpart. Both enzymes showed pre-transfer editing activity implying their high overall accuracy. The contribution of various reaction pathways in the pre-transfer editing reactions by maize enzymes were different and dependent on the non-cognate substrate. The fidelity mechanisms of maize organellar SerRS, high discriminatory power and proofreading, indicate that aaRSs in general may play an important role in translational quality control in plant mitochondria and chloroplasts.

Evolutionarily conserved cysteines in plant cytosolic seryl-tRNA synthetase are important for its resistance to oxidation.

We have previously identified a unique disulfide bond in the crystal structure of Arabidopsis cytosolic seryl-tRNA synthetase involving cysteines evolutionarily conserved in all green plants. Here, we discovered that both cysteines are important for protein stability, but with opposite effects, and that their microenvironment may promote disulfide bond formation in oxidizing conditions. The crystal structure of the C244S mutant exhibited higher rigidity and an extensive network of noncovalent interactions correlating with its higher thermal stability. The activity of the wild-type showed resistance to oxidation with H2 O2 , while the activities of cysteine-to-serine mutants were impaired, indicating that the disulfide link may enable the protein to function under oxidative stress conditions which can be beneficial for an efficient plant stress response.

Importance of protein intrinsic conformational dynamics and transient nature of non-covalent interactions in ligand binding affinity.

We have recently identified BEN1 as a protein interactor of seryl-tRNA synthetase (SerRS) from model plant Arabidopsis thaliana. BEN1 contains an NADP+ binding domain and possesses acidic N-terminal extension essential for interaction with A. thaliana SerRS. This extension, specific for BEN1 homologues from Brassicaceae family, is solvent-exposed and distant to the nucleotide-binding site. We prepared a truncated BEN1 variant ΔN17BEN1 lacking the first 17 amino acid of this N-terminal extension as well as full-length BEN1 to investigate how the truncation affects the binding affinity towards coenzyme NADP+. By performing microscale thermophoresis (MST) experiments we have shown that both BEN1 variants bind the NADP+ cofactor, however, truncated BEN1 showed 34-fold higher affinity towards NADP+ indicating that its core protein structure is not just preserved but it binds NADP+ even stronger. To further corroborate the obtained results, we opted for a computational approach based on classical molecular dynamics simulations of both complexes. Our results have shown that both truncated and intact BEN1 variants form the same number of interactions with the NADP+ cofactor; however, it was the interaction occupancy that was affected. Namely, three independent MD simulations showed that the ΔN17BEN1 variant in complex with NADP+ has significantly higher interaction occupancy thus binds NADP+ with more than one order of magnitude higher affinity. Contrary to our expectations, the truncation of this distant region that does not communicate with the nucleotide-binding site didn't result in the gain of interaction but affected the intrinsic conformational dynamics which in turn fine-tuned the binding affinity by increasing the interaction occupancy and strength of the key conserved cation-π interaction between Arg69 and adenine of NADP+ and hydrogen bond between Ser244 and phosphate of NADP+.

Arabidopsis seryl-tRNA synthetase: the first crystal structure and novel protein interactor of plant aminoacyl-tRNA synthetase.

The rules of the genetic code are established by aminoacyl-tRNA synthetases (aaRSs) enzymes, which covalently link tRNA with the cognate amino acid. Many aaRSs are involved in diverse cellular processes beyond translation, acting alone, or in complex with other proteins. However, studies of aaRS noncanonical assembly and functions in plants are scarce, as are structural studies of plant aaRSs. Here, we have solved the crystal structure of Arabidopsis thaliana cytosolic seryl-tRNA synthetase (SerRS), which is the first crystallographic structure of a plant aaRS. Arabidopsis SerRS displays structural features typical of canonical SerRSs, except for a unique intrasubunit disulfide bridge. In a yeast two-hybrid screen, we identified BEN1, a protein involved in the metabolism of plant brassinosteroid hormones, as a protein interactor of Arabidopsis SerRS. The SerRS:BEN1 complex is one of the first protein complexes of plant aaRSs discovered so far, and is a rare example of an aaRS interacting with an enzyme involved in primary or secondary metabolism. To pinpoint regions responsible for this interaction, we created truncated variants of SerRS and BEN1, and identified that the interaction interface involves the SerRS globular catalytic domain and the N-terminal extension of BEN1 protein. BEN1 does not have a strong impact on SerRS aminoacylation activity, indicating that the primary function of the complex is not the modification of SerRS canonical activity. Perhaps SerRS performs as yet unknown noncanonical functions mediated by BEN1. These findings indicate that - via SerRS and BEN1 - a link exists between the protein translation and steroid metabolic pathways of the plant cell. DATABASE: Structural data are available in the PDB under the accession number PDB ID 6GIR.

Structural flexibility of the methanogenic-type seryl-tRNA synthetase active site and its implication for specific substrate recognition.

Seryl-tRNA synthetase (SerRS) is a class II aminoacyl-tRNA synthetase that catalyzes serine activation and its transfer to cognate tRNA(Ser). Previous biochemical and structural studies have revealed that bacterial- and methanogenic-type SerRSs employ different strategies of substrate recognition. In addition to other idiosyncratic features, such as the active site zinc ion and the unique fold of the N-terminal tRNA-binding domain, methanogenic-type SerRS is, in comparison with bacterial homologues, characterized by a notable shortening of the motif 2 loop. Mutational analysis of Methanosarcina barkeri SerRS (mMbSerRS) was undertaken to identify the active site residues that ensure the specificity of amino acid and tRNA 3'-end recognition. Residues predicted to contribute to the amino acid specificity were selected for mutation according to the crystal structure of mMbSerRS complexed with its cognate aminoacyl-adenylate, whereas those involved in binding of the tRNA 3'-end were identified and mutagenized on the basis of modeling the mMbSerRS:tRNA complex. Although mMbSerRSs variants with an altered serine-binding pocket (W396A, N435A, S437A) were more sensitive to inhibition by threonine and cysteine, none of the mutants was able to activate noncognate amino acids to greater extent than the wild-type enzyme. In vitro kinetics results also suggest that conformational changes in the motif 2 loop are required for efficient serylation.

Hydrolysis of non-cognate aminoacyl-adenylates by a class II aminoacyl-tRNA synthetase lacking an editing domain.

Aminoacyl-tRNA synthetases, a group of enzymes catalyzing aminoacyl-tRNA formation, may possess inherent editing activity to clear mistakes arising through the selection of non-cognate amino acid. It is generally assumed that both editing substrates, non-cognate aminoacyl-adenylate and misacylated tRNA, are hydrolyzed at the same editing domain, distant from the active site. Here, we present the first example of an aminoacyl-tRNA synthetase (seryl-tRNA synthetase) that naturally lacks an editing domain, but possesses a hydrolytic activity toward non-cognate aminoacyl-adenylates. Our data reveal that tRNA-independent pre-transfer editing may proceed within the enzyme active site without shuttling the non-cognate aminoacyl-adenylate intermediate to the remote editing site.

Dual targeting of organellar seryl-tRNA synthetase to maize mitochondria and chloroplasts.

Aminoacyl-tRNA synthetases (AARSs) play a critical role in translation and are thus required in three plant protein-synthesizing compartments: cytosol, mitochondria and plastids. A systematic study had previously shown extensive sharing of organellar AARSs from Arabidopsis thaliana, mostly between mitochondria and chloroplasts. However, distribution of AARSs from monocot species, such as maize, has never been experimentally investigated. Here we demonstrate dual targeting of maize seryl-tRNA synthetase, SerZMo, into both mitochondria and chloroplasts using combination of complementary methods, including in vitro import assay, transient expression analysis of green fluorescent protein (GFP) fusions and immunodetection. We also show that SerZMo dual localization is established by the virtue of an ambiguous targeting peptide. Full-length SerZMo protein fused to GFP is targeted to chloroplast stromules, indicating that SerZMo protein performs its function in plastid stroma. The deletion mutant lacking N-terminal region of the ambiguous SerZMo targeting peptide was neither targeted into mitochondria nor chloroplasts, indicating the importance of this region in both mitochondrial and chloroplastic import.

Maize seryl-tRNA synthetase: specificity of substrate recognition by the organellar enzyme.

In our study of seryl-tRNA formation in maize, we investigated the enzymes involved in serylation. Only two dissimilar seryl-tRNA synthetase (SerRS) cDNA clones were identified in the Zea mays EST (expressed sequence tag) databases. One encodes a seryl-tRNA synthetase, which presumably functions in the organelles (SerZMm), while the other synthetase product is more similar to eukaryotic cytosolic counterparts (SerZMc). The expression of SerZMm in Saccharomyces cerevisiae resulted in complementation of mutant respiratory phenotype, caused by a disruption of the nuclear gene, presumably encoding yeast mitochondrial seryl-tRNA synthetase (SerSCm). Purified mature SerZMm displays tRNA-assisted serine activation and aminoacylates maize mitochondrial and chloroplast tRNA(Ser) transcripts with similar efficiencies, raising the possibility that only two isoforms of seryl-tRNA synthetase may be sufficient to catalyze seryl-tRNA(Ser) formation in three cellular compartments of Zea mays. Phylogenetic analysis suggests that SerZMm is of mitochondrial origin.