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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. All authors acknowledge the COST Action project ARBRE-MOBIEU CA15126 under the auspices of whose Working Group 4 this study was carried out. ARBRE-MOBIEU COST Action CA15126 funded meetings in the Center for Protein Research, University of Copenhagen (ECOST-MEETING-CA15126-280618-098884) to organize this study and in the Vienna Biocenter Core Facilities GmbH (ECOST-MEETING-CA15126-121119-111544) to discuss the results of the benchmark. Ondřej Vaněk acknowledges support from the Ministry of Education, Youth and Sports of the Czech Republic (LTC17065 in frame of the COST Action CA15126). Jitka Holková and Josef Houser acknowledge CF Biomolecular Interactions and Crystallization of CIISB, Instruct-CZ Centre, supported by MEYS CR (LM2018127). Jasmina Rokov-Plavec acknowledges support from the Croatian Science Foundation (IP-2016-06-6272). Alexander K. Buell thanks the Novo Nordisk Foundation (grant number NNFSA170028392) for funding. Li Peng Lundgren acknowledges for preliminary MST/TRIC experiments (STMS 37745 in the frame of the COST Action CA15126) and the COST Action (CA15126) for a STMS (STMS 44783) for the data analysis. The Novo Nordisk Foundation Center for Protein Research is supported financially by the Novo Nordisk Foundation (Grant agreement NNF14CC0001). Katja Pirc and Marjetka Podobnik acknowledge the grant by the Slovenian Research Agency P1-0391 (Title: Molecular Interactions). This research was funded by Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III and European Union (ERDF/ESF, “Investing in your future” PI18/00349 and Diputación General de Aragón Digestive Pathology Group B25_17R to O.A. Spanish Ministry of Science, Innovation and Universities, FPI Predoctoral Research Contract BES-2017-080739 to D.O.A. This research was funded by project no 2015/17/B/NZ2/01160 (granted to MA)

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+.

For a long time seryl-tRNA synthetases (SerRSs) stood as an archetypal, canonical aminoacyl-tRNA synthetases (aaRS), exhibiting only basic tRNA aminoacylation activity and with no moonlighting functions beyond protein biosynthesis. The picture has changed substantially in recent years after the discovery that SerRSs play an important role in antibiotic production and resistance and act as a regulatory factor in vascular development, as well as after the discovery of mitochondrial morphogenesis factor homologous to SerRS in insects. In this review we summarize the recent research results from our laboratory, which advance the understanding of seryl-tRNA synthetases and further paint the dynamic picture of unexpected SerRS activities. SerRS from archaeon Methanothermobacter thermautotrophicus was shown to interact with the large ribosomal subunit and it was postulated to contribute to a more efficient translation by the"tRNA channeling" hypothesis. Discovery of the atypical SerRS in a small number of methanogenic archaea led to the discovery of a new family of enzymes in numerous bacteria - amino acid:[carrier protein] ligases (aa:CP ligases). These SerRS homologues resigned tRNA aminoacylation activity, and instead adopted carrier proteins as the acceptors of activated amino acids. The crystal structure of the aa:CP ligase complex with the carrier protein revealed that the interactions between two macromolecules are incomparable to tRNA binding by the aaRS and consequently represent a true evolutionary invention. Kinetic investigations of SerRSs and the accuracy of amino acid selection revealed that SerRSs possess pre-transfer proofreading activity, challenging the widely accepted presumption that hydrolytic proofreading activity must reside in an additional, separate editing domain, not present in SerRSs. Finally, the plant tRNA serylation system is discussed, which is particularly interesting due to the fact that protein biosynthesis takes place in three cellular compartments: cytosol, mitochondria and chloroplasts. Plant cytosolic SerRSs showed broad tRNASer specificity and flexibility, unlike SerRSs from other organisms. High fidelity of SerRS dually targeted to mitochondria and chloroplasts indicated its importance in plant organellar quality control. This work is licensed under a Creative Commons Attribution 4.0 International License.

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.

Aminoacyl-tRNA synthetases (AARS) are essential enzymes that covalently link amino acid and tRNA. Aminoacylated tRNAs are substrates for protein translation on ribosomes. Besides their central role in translation, AARSs from various organisms participate in other essential cellular processes. However, there are only two reports on noncanonical functions of plant AARSs. Arabidopsis aspartyl-tRNA synthetase and rice methionyl-tRNA synthetase are involved in plant defense mechanisms against pathogen attack. In order to reveal novel noncanonical functions of plant AARSs, we determined the expression levels of selected AARS genes in Arabidopsis thaliana seedlings exposed to various abiotic stress conditions (high heavy metal concentration (cadmium), salinity and osmotic stress) using RT- qPCR. In another set of experiments, we analyzed two transgenic A. thaliana lines. The transgenic lines contained a transgene consisting of coding sequences for seryl-tRNA synthetase (SerRS) and a tag (either GFP or TAP) under regulation of a constitutive promoter. Using RT-qPCR we determined overexpression levels of SerRS transgenes. Western blot analysis and aminoacylation assays showed that the fusion SerRS proteins are active. The two transgenic lines and wild type A. thaliana seedlings were grown on medium containing different stressors (cadmium, salt, and mannitol) and the length of the roots was monitored for several days. Our results indicate that AARSs could have a role in cellular response to abiotic stress in plants.

Seryl-tRNA synthetase (SerRS) is a class II aminoacyl-tRNA synthetase that catalyzes serine activation and its transfer to cognate tRNASer. 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.

Aminoacyl-tRNA synthetases (aaRSs) decipher the genetic code, covalently linking amino acids to cognate tRNAs, thus preparing substrates for the process of translation. Although aaRSs funtion primarily in translation and are localized in cytosol, mitochondria and chloroplasts there are many reports on their additional functions and subcellular destinations beyond translation. However, data on plant aaRSs are scarce. Initial analysis of amino acid sequence of Arabidopsis thaliana seryl-tRNA synthetase (SerRS) suggested that protein contains putative nuclear localization signals. GFP-localization experiments in transiently transformed epidermal onion cells and Arabidopsis protoplasts gave ambiguous results because in some cells SerRS appeared to be dually localized to both cytosol and nucleus. However, data obtained on transgenic lines expressing SerRS-TAP and GFP-SerRS revealed exclusive cytosolic location of SerRS. Subcellular distribution of SerRS did not change during stress. Cytosolic Arabidopsis SerRS was expressed and purified. The enzyme efficiently aminoacylated eukaryotic and bacterial tRNAsSer, that are structurally very different. Given the fact that the same behavior was previously shown for monocot maize SerRS, it seems that plant SerRSs exhibit unusually broad tRNASer specificity, unlike SerRSs from other organisms. Possible functional implications of this unique characteristic of plant SerRSs are discussed.

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.

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.

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.

Noncovalent interactions of yeast homodimeric seryl-tRNA synthetase (SerRS) and cognate tRNA(Ser) were studied by the gel mobility shift assay and zone-interference gel electrophoresis performed under the same binding and electrophoretic conditions. Purified tRNA(Ser) as well as total yeast tRNA were applied as ligands. In the absence of Mg2+ SerRS:(tRNA(Ser))(1) noncovalent complex was detected only by zone-interference gel electrophoresis. K-d values determined in the presence and absence of Mg2+ were in the same range, suggesting that Mg2+ ions mainly influence dissociation-association kinetics of the complex, with a minor contribution to its thermodynamic stability. Comparison of these two assays was shown to be useful in the analysis of thermodynamic and kinetic properties of protein:nucleic acid complexes.

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.

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.