Free Energy Landscape of the Adenylation Reaction of the Aminoacylation Process at the Active Site of Aspartyl tRNA Synthetase.

The process of protein biosynthesis is initiated by the aminoacylation process where a transfer ribonucleic acid (tRNA) is charged by the attachment of its cognate amino acid at the active site of the corresponding aminoacyl tRNA synthetase enzyme. The first step of the aminoacylation process, known as the adenylation reaction, involves activation of the cognate amino acid where it reacts with a molecule of adenosine triphosphate (ATP) at the active site of the enzyme to form the aminoacyl adenylate and inorganic pyrophosphate. In the current work, we have investigated the adenylation reaction between aspartic acid and ATP at the active site of the fully solvated aspartyl tRNA synthetase (AspRS) from Escherichia coli in aqueous medium at room temperature through hybrid quantum mechanical/molecular mechanical (QM/MM) simulations combined with enhanced sampling methods of well-tempered and well-sliced metadynamics. The objective of the present work is to study the associated free energy landscape and reaction barrier and also to explore the effects of active site mutation on the free energy surface of the reaction. The current calculations include finite temperature effects on free energy profiles. In particular, apart from contributions of interaction energies, the current calculations also include contributions of conformational, vibrational, and translational entropy of active site residues, substrates, and also the rest of the solvated protein and surrounding water into the free energy calculations. The present QM/MM metadynamics simulations predict a free energy barrier of 23.35 and 23.5 kcal/mol for two different metadynamics methods used to perform the reaction at the active site of the wild type enzyme. The free energy barrier increases to 30.6 kcal/mol when Arg217, which is an important conserved residue of the wild type enzyme at its active site, is mutated by alanine. These free energy results including the effect of mutation compare reasonably well with those of kinetic experiments that are available in the literature. The current work also provides molecular details of structural changes of the reactants and surroundings as the system dynamically evolves along the reaction pathway from reactant to the product state through QM/MM metadynamics simulations.

A single amino acid substitution alters activity and specificity in Plasmodium falciparum aspartyl & asparaginyl-tRNA synthetases.

The specificity of each aminoacyl-tRNA synthetase (aaRS) for its cognate amino acid ensures correct tRNA esterification and allows fidelity in protein synthesis. The aaRSs discriminate based on the chemical properties of their amino acid substrates and structural features of the binding pockets. In this study, we characterized aspartyl-(DRS) and asparaginyl-tRNA synthetase (NRS) from Plasmodium falciparum to determine the basis of their specificity towards L-asp and L-asn respectively. The negatively charged L-asp and its analogue L-asn differ only in their side-chain groups i.e., -OH and -NH2. Further, the amino acid binding sites are highly conserved within these two enzymes. Analysis of the substrate (L-asp/L-asn) binding sites across species revealed two highly conserved residues in PfDRS (D408 and K372) and PfNRS (E395 and L360) that are involved in recognition of the Oδ2/Nδ2 of L-asp/L-asn respectively. These residues were mutated and swapped between the D408→E in PfDRS and the corresponding E395→D in PfNRS. A similar approach was employed for residue number K372→L in PfDRS and L360→K in PfNRS. The mutated PfDRSD408E retained its enzymatic activity during step 1 of aminoacylation reaction towards L-asp and L-asn and esterified tRNAAsp with L-asp like wild type enzyme, while the PfDRSK372L was rendered enzymatically inactive. The correspondingly mutated PfNRSE395D was enzymatically inactive. The mutated PfNRSL360K had an altered specificity and esterified tRNAAsn with non-cognate amino acid L-asp and not L-asn. These data suggest that the residue K372 is crucial for the enzymatic activity of PfDRS while the residue L360 in PfNRS imparts specificity towards L-asn.

Anti-HIV Effects of Baculiferins Are Regulated by the Potential Target Protein DARS.

Baculiferins are a group of marine sponge-derived polycyclic alkaloids with anti-HIV (human immunodeficiency virus) activities. To identify additional baculiferin-based congeners for SAR analysis and to investigate the mode of action, a total of 18 new baculiferin-type derivatives were synthesized. The inhibitory activities of the congeners against the HIV-1 virus were evaluated in vitro, and the relevant SAR was discussed. Compound 18 exerted the most potent activity toward VSV-G-pseudotyped HIV-1 (IC50 of 3.44 µM) and HIV-1 strain SF33 (IC50 of 2.80 µM) in vitro. To identify the cellular targets, three photoaffinity baculiferin probes were simultaneously synthesized. Photoaffinity labeling experiments together with LC-MS/MS data identified aspartate-tRNA ligase (DARS) as a putative target protein of 18. The overexpression and knockdown of DARS in HEK293T cells provided additional data to demonstrate that DARS is a potential target protein in the regulation of HIV virus infection. The modes of antiviral baculiferins 13 and 18 binding to DARS were determined by a molecular docking simulation. Thus, baculiferin 18 is considered a promising lead as a new molecular target for the development of anti-HIV agents.

Anticodon-binding domain swapping in a nondiscriminating aspartyl-tRNA synthetase reveals contributions to tRNA specificity and catalytic activity.

The nondiscriminating aspartyl-tRNA synthetase (ND-AspRS), found in many archaea and bacteria, covalently attaches aspartic acid to tRNAAsp and tRNAAsn generating a correctly charged Asp-tRNAAsp and an erroneous Asp-tRNAAsn . This relaxed tRNA specificity is governed by interactions between the tRNA and the enzyme. In an effort to assess the contributions of the anticodon-binding domain to tRNA specificity, we constructed two chimeric enzymes, Chimera-D and Chimera-N, by replacing the native anticodon-binding domain in the Helicobacter pylori ND-AspRS with that of a discriminating AspRS (Chimera-D) and an asparaginyl-tRNA synthetase (AsnRS, Chimera-N), both from Escherichia coli. Both chimeric enzymes showed similar secondary structure compared to wild-type (WT) ND-AspRS and maintained the ability to form dimeric complexes in solution. Although less catalytically active than WT, Chimera-D was more discriminating as it aspartylated tRNAAsp over tRNAAsn with a specificity ratio of 7.0 compared to 2.9 for the WT enzyme. In contrast, Chimera-N exhibited low catalytic activity toward tRNAAsp and was unable to aspartylate tRNAAsn . The observed catalytic activities for the two chimeras correlate with their heterologous toxicity when expressed in E. coli. Molecular dynamics simulations show a reduced hydrogen bond network at the interface between the anticodon-binding domain and the catalytic domain in Chimera-N compared to Chimera-D or WT, explaining its lower stability and catalytic activity.

Leishmanial aspartyl-tRNA synthetase: Biochemical, biophysical and structural insights.

Aminoacyl tRNA synthetases (aaRSs) are integral components of protein biosynthesis along with several non-canonical cellular processes. Inhibition studies of aaRSs presented these enzymes as promising drug targets in many pathogens, however aspartyl tRNA synthetase has not been studied in trypanosomatids despite its essentiality. Hence, full-length ORF of Leishmania donovani aspartyl tRNA synthetase (LdaspRS) was cloned and purified to homogeneity followed by molecular mass determination. The aminoacylation assay established that the purified protein performs its function optimally at physiological pH and temperature. The kinetic parameters of LdaspRS revealed the affinity of l-aspartate towards the enzyme to be very much lower than the cofactor. Our study also highlights the moonlighting function of LdaspRS to stimulate the pro-inflammatory cytokines and nitric oxide generation by host macrophage. Furthermore, CD and intrinsic tryptophan fluorescence measurements showed the changes in structural conformation at varying pH, denaturants and ligands. The modelled LdaspRS structure presented all the specific characteristics of class II aaRSs, while in silico study suggested binding of pyrimidine-derived inhibitors in its cofactor binding site with high affinity followed by validation using MD simulation. Altogether, this study could provide a platform for exploring LdaspRS to develop potential therapeutics against leishmaniasis.

Symmetric Assembly of a Decameric Subcomplex in Human Multi-tRNA Synthetase Complex Via Interactions between Glutathione Transferase-Homology Domains and Aspartyl-tRNA Synthetase.

Aminoacyl-tRNA synthetases (AARSs) ligate amino acids to their cognate tRNAs during protein synthesis. In humans, eight AARSs and three non-enzymatic AARS-interacting multifunctional proteins (AIMP1-3), which are involved in various biological processes, form a multi-tRNA synthetase complex (MSC). Elucidation of the structures and multiple functions of individual AARSs and AIMPs has aided current understanding of the structural arrangement of MSC components and their assembly processes. Here, we report the crystal structure of a complex comprising a motif from aspartyl-tRNA synthetase (DRS) and the glutathione transferase (GST)-homology domains of methionyl-tRNA synthetase (MRS), glutamyl-prolyl-tRNA synthetase (EPRS), AIMP2, and AIMP3. In the crystal structure, the four GST domains are assembled in the order of MRS-AIMP3-EPRS-AIMP2, and the GST domain of AIMP2 binds DRS through the ß-sheet in the GST domain. The C-terminus of AIMP3 enhances the binding of DRS to the tetrameric GST complex. A DRS dimer and two GST tetramers binding to the dimer with 2-fold symmetry complete a decameric complex. The formation of this complex enhances the stability of DRS and enables it to retain its reaction intermediate, aspartyl adenylate. Since the catalytic domains of MRS and EPRS are connected to the decameric complex through their flexible linker peptides, and lysyl-tRNA synthetase and AIMP1 are also linked to the complex via the N-terminal region of AIMP2, the DRS-GST tetramer complex functions as a frame in the MSC.

Bacterial Aspartyl-tRNA Synthetase Has Glutamyl-tRNA Synthetase Activity.

The aminoacyl-tRNA synthetases (aaRSs) are well established as the translators of the genetic code, because their products, the aminoacyl-tRNAs, read codons to translate messenger RNAs into proteins. Consequently, deleterious errors by the aaRSs can be transferred into the proteome via misacylated tRNAs. Nevertheless, many microorganisms use an indirect pathway to produce Asn-tRNAAsn via Asp-tRNAAsn. This intermediate is produced by a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) that has retained its ability to also generate Asp-tRNAAsp. Here we report the discovery that ND-AspRS and its discriminating counterpart, AspRS, are also capable of specifically producing Glu-tRNAGlu, without producing misacylated tRNAs like Glu-tRNAAsn, Glu-tRNAAsp, or Asp-tRNAGlu, thus maintaining the fidelity of the genetic code. Consequently, bacterial AspRSs have glutamyl-tRNA synthetase-like activity that does not contaminate the proteome via amino acid misincorporation.

Spectroscopic Studies of Asparaginyl-tRNA Synthetase from Entamoeba histolytica.

BACKGROUND: Aminoacyl-tRNA synthetases play an important role in catalyzing the first step in protein synthesis by attaching the appropriate amino acid to its cognate tRNA which then transported to the growing polypeptide chain. Asparaginyl-tRNA Synthetase (AsnRS) from Brugia malayi, Leishmania major, Thermus thermophilus, Trypanosoma brucei have been shown to play an important role in survival and pathogenesis. Entamoeba histolytica (Ehis) is an anaerobic eukaryotic pathogen that infects the large intestines of humans. It is a major cause of dysentery and has the potential to cause life-threatening abscesses in the liver and other organs making it the second leading cause of parasitic death after malaria. Ehis-AsnRS has not been studied in detail, except the crystal structure determined at 3 Å resolution showing that it is primarily α-helical and dimeric. It is a homodimer, with each 52 kDa monomer consisting of 451 amino acids. It has a relatively short N-terminal as compared to its human and yeast counterparts. OBJECTIVE: Our study focusses to understand certain structural characteristics of Ehis-AsnRS using biophysical tools to decipher the thermodynamics of unfolding and its binding properties. METHODS: Ehis-AsnRS was cloned and expressed in E. coli BL21DE3 cells. Protein purification was performed using Ni-NTA affinity chromatography, following which the protein was used for biophysical studies. Various techniques such as steady-state fluorescence, quenching, circular dichroism, differential scanning fluorimetry, isothermal calorimetry and fluorescence lifetime studies were employed for the conformational characterization of Ehis-AsnRS. Protein concentration for far-UV and near-UV circular dichroism experiments was 8 µM and 20 µM respectively, while 4 µM protein was used for the rest of the experiments. RESULTS: The present study revealed that Ehis-AsnRS undergoes unfolding when subjected to increasing concentration of GdnHCl and the process is reversible. With increasing temperature, it retains its structural compactness up to 45ºC before it unfolds. Steady-state fluorescence, circular dichroism and hydrophobic dye binding experiments cumulatively suggest that Ehis-AsnRS undergoes a two-state transition during unfolding. Shifting of the transition mid-point with increasing protein concentration further illustrate that dissociation and unfolding processes are coupled indicating the absence of any detectable folded monomer. CONCLUSION: This article indicates that GdnHCl induced denaturation of Ehis-AsnRS is a two - state process and does not involve any intermediate; unfolding occurs directly from native dimer to unfolded monomer. The solvent exposure of the tryptophan residues is biphasic, indicating selective quenching. Ehis-AsnRS also exhibits a structural as well as functional stability over a wide range of pH.

Structure-function studies of the asparaginyl-tRNA synthetase from Fasciola gigantica: understanding the role of catalytic and non-catalytic domains.

The asparaginyl-tRNA synthetase (NRS) catalyzes the attachment of asparagine to its cognate tRNA during translation. NRS first catalyzes the binding of Asn and ATP to form the NRS-asparaginyl adenylate complex, followed by the esterification of Asn to its tRNA. We investigated the role of constituent domains in regulating the structure and activity of Fasciola gigantica NRS (FgNRS). We cloned the full-length FgNRS, along with its various truncated forms, expressed, and purified the corresponding proteins. Size exclusion chromatography indicated a role of the anticodon-binding domain (ABD) of FgNRS in protein dimerization. The N-terminal domain (NTD) was not essential for cognate tRNA binding, and the hinge region between the ABD and the C-terminal domain (CTD) was crucial for regulating the enzymatic activity. Molecular docking and fluorescence quenching experiments elucidated the binding affinities of the substrates to various domains. The molecular dynamics simulation of the modeled protein showed the presence of an unstructured region between the NTD and ABD that exhibited a large number of conformations over time, and further analysis indicated this region to be intrinsically disordered. The present study provides information on the structural and functional regulation, protein-substrate(s) interactions and dynamics, and the role of non-catalytic domains in regulating the activity of FgNRS.

Unique N-terminal extension domain of human asparaginyl-tRNA synthetase elicits CCR3-mediated chemokine activity.

Asparaginyl-tRNA synthetase (NRS) is not only essential in protein translation but also associated with autoimmune diseases. Particularly, patients with antibodies that recognize NRS often develop interstitial lung disease (ILD). However, the underlying mechanism of how NRS is recognized by immune cells and provokes inflammatory responses is not well-understood. Here, we found that the crystal structure of the unique N-terminal extension domain of human NRS (named as UNE-N, where -N denotes NRS) resembles that of the chemotactic N-terminal domain of NRS from a filarial nematode, Brugia malayi, which recruits and activates specific immune cells by interacting with CXC chemokine receptor 1 and 2. UNE-N induced migration of CC chemokine receptor 3 (CCR3)-expressing cells. The chemokine activity of UNE-N was significantly reduced by suppressing CCR3 expression with CCR3-targeting siRNA, and the loop3 region of UNE-N was shown to interact mainly with the extracellular domains of CCR3 in nuclear magnetic resonance perturbation experiments. Based on these results, evolutionarily acquired UNE-N elicits chemokine activities that would promote NRS-CCR3-mediated proinflammatory signaling in ILD.

Soluble expression and purification of a full-length asparaginyl tRNA synthetase from Fasciola gigantica.

We report the molecular cloning, expression, and single-step homogeneous purification of a full-length asparaginyl tRNA synthetase (NRS) from Fasciola gigantica (FgNRS). Fasciola gigantica is a parasitic liver fluke of the class Trematoda. It causes fascioliasis that infects the liver of various mammals, including humans. Aminoacyl tRNA synthetases (AARS) catalyze the first step of protein synthesis. They attach an amino acid to its cognate tRNA, forming an amino acid-tRNA complex. The gene that codes for FgNRS was generated by amplification by polymerase chain reaction. It was then inserted in the expression vector pQE30 under the transcriptional control of the bacteriophage T5 promoter and lac operator. M15 Escherichia coli strain transformed with the FgNRS expression vector pQE30-NRS accumulates large amounts of a soluble protein of about 61 kDa. The protein was purified to homogeneity using immobilized metal affinity chromatography. The recombinant protein was further confirmed by immunoblotting with anti-His antibody. Following size exclusion chromatography, the FgNRS was stable and observed to be a dimeric protein. In this study, the expression and purification procedures have provided a simple and efficient method to obtain full-length FgNRS in large quantities. This will provide an opportunity to study the structure, dynamics and function of NRS.

Crystal structure of the N-terminal anticodon-binding domain of the nondiscriminating aspartyl-tRNA synthetase from Helicobacter pylori.

The N-terminal anticodon-binding domain of the nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) plays a crucial role in the recognition of both tRNAAsp and tRNAAsn. Here, the first X-ray crystal structure of the N-terminal domain of this enzyme (ND-AspRS1-104) from the human-pathogenic bacterium Helicobacter pylori is reported at 2.0 Šresolution. The apo form of H. pylori ND-AspRS1-104 shares high structural similarity with the N-terminal anticodon-binding domains of the discriminating aspartyl-tRNA synthetase (D-AspRS) from Escherichia coli and ND-AspRS from Pseudomonas aeruginosa, allowing recognition elements to be proposed for tRNAAsp and tRNAAsn. It is proposed that a long loop (Arg77-Lys90) in this H. pylori domain influences its relaxed tRNA specificity, such that it is classified as nondiscriminating. A structural comparison between D-AspRS from E. coli and ND-AspRS from P. aeruginosa suggests that turns E and F (78GAGL81 and 83NPKL86) in H. pylori ND-AspRS play a crucial role in anticodon recognition. Accordingly, the conserved Pro84 in turn F facilitates the recognition of the anticodons of tRNAAsp (34GUC36) and tRNAAsn (34GUU36). The absence of the amide H atom allows both C and U bases to be accommodated in the tRNA-recognition site.

A new application of click chemistry in situ: development of fluorescent probe for specific G-quadruplex topology.

Target-guided synthesis is an approach to drug discovery that allows the target to self-assemble its own binding agents. So far, target-guided synthesis and especially in situ click chemistry have attracted extensive attention and have led to the identification of highly potent inhibitors for proteins. In this study, we expand the application of in situ click chemistry and present a procedure using this approach to identify selective fluorescent probes for a specific topology of G-quadruplex nucleic acids, the parallel G-quadruplexes. On this basis, compound 15 assembled by triarylimidazole scaffold and carboxyl side chain was a positive hit, demonstrating highly potential in the sensitive and selective detection of parallel G-quadruplexes. Such selective fluorescence response can be rationalized in terms of different binding affinities between 15 and G-quadruplexes. Our work accordingly represents a new development towards the application of in situ click chemistry to develop selective fluorescent probes and may also shed light on the search for probes for a specific G-quadruplex topology.

Structure of the Pseudomonas aeruginosa transamidosome reveals unique aspects of bacterial tRNA-dependent asparagine biosynthesis.

Many prokaryotes lack a tRNA synthetase to attach asparagine to its cognate tRNA(Asn), and instead synthesize asparagine from tRNA(Asn)-bound aspartate. This conversion involves two enzymes: a nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) that forms Asp-tRNA(Asn), and a heterotrimeric amidotransferase GatCAB that amidates Asp-tRNA(Asn) to form Asn-tRNA(Asn) for use in protein synthesis. ND-AspRS, GatCAB, and tRNA(Asn) may assemble in an ∼400-kDa complex, known as the Asn-transamidosome, which couples the two steps of asparagine biosynthesis in space and time to yield Asn-tRNA(Asn). We report the 3.7-Šresolution crystal structure of the Pseudomonas aeruginosa Asn-transamidosome, which represents the most common machinery for asparagine biosynthesis in bacteria. We show that, in contrast to a previously described archaeal-type transamidosome, a bacteria-specific GAD domain of ND-AspRS provokes a principally new architecture of the complex. Both tRNA(Asn) molecules in the transamidosome simultaneously serve as substrates and scaffolds for the complex assembly. This architecture rationalizes an elevated dynamic and a greater turnover of ND-AspRS within bacterial-type transamidosomes, and possibly may explain a different evolutionary pathway of GatCAB in organisms with bacterial-type vs. archaeal-type Asn-transamidosomes. Importantly, because the two-step pathway for Asn-tRNA(Asn) formation evolutionarily preceded the direct attachment of Asn to tRNA(Asn), our structure also may reflect the mechanism by which asparagine was initially added to the genetic code.

Biochemical and structural characterization of mycobacterial aspartyl-tRNA synthetase AspS, a promising TB drug target.

The human pathogen Mycobacterium tuberculosis is the causative agent of pulmonary tuberculosis (TB), a disease with high worldwide mortality rates. Current treatment programs are under significant threat from multi-drug and extensively-drug resistant strains of M. tuberculosis, and it is essential to identify new inhibitors and their targets. We generated spontaneous resistant mutants in Mycobacterium bovis BCG in the presence of 10× the minimum inhibitory concentration (MIC) of compound 1, a previously identified potent inhibitor of mycobacterial growth in culture. Whole genome sequencing of two resistant mutants revealed in one case a single nucleotide polymorphism in the gene aspS at (535)GAC>(535)AAC (D179N), while in the second mutant a single nucleotide polymorphism was identified upstream of the aspS promoter region. We probed whole cell target engagement by overexpressing either M. bovis BCG aspS or Mycobacterium smegmatis aspS, which resulted in a ten-fold and greater than ten-fold increase, respectively, of the MIC against compound 1. To analyse the impact of inhibitor 1 on M. tuberculosis AspS (Mt-AspS) activity we over-expressed, purified and characterised the kinetics of this enzyme using a robust tRNA-independent assay adapted to a high-throughput screening format. Finally, to aid hit-to-lead optimization, the crystal structure of apo M. smegmatis AspS was determined to a resolution of 2.4 Å.

The predatory bacterium Bdellovibrio bacteriovorus aspartyl-tRNA synthetase recognizes tRNAAsn as a substrate.

The predatory bacterium Bdellovibrio bacteriovorus preys on other Gram-negative bacteria and was predicted to be an asparagine auxotroph. However, despite encoding asparaginyl-tRNA synthetase and glutaminyl-tRNA synthetase, B. bacteriovorus also contains the amidotransferase GatCAB. Deinococcus radiodurans, and Thermus thermophilus also encode both of these aminoacyl-tRNA synthetases with GatCAB. Both also code for a second aspartyl-tRNA synthetase and use the additional aspartyl-tRNA synthetase with GatCAB to synthesize asparagine on tRNAAsn. Unlike those two bacteria, B. bacteriovorus encodes only one aspartyl-tRNA synthetase. Here we demonstrate the lone B. bacteriovorus aspartyl-tRNA synthetase catalyzes aspartyl-tRNAAsn formation that GatCAB can then amidate to asparaginyl-tRNAAsn. This non-discriminating aspartyl-tRNA synthetase with GatCAB thus provides B. bacteriovorus a second route for Asn-tRNAAsn formation with the asparagine synthesized in a tRNA-dependent manner. Thus, in contrast to a previous prediction, B. bacteriovorus codes for a biosynthetic route for asparagine. Analysis of bacterial genomes suggests a significant number of other bacteria may also code for both routes for Asn-tRNAAsn synthesis with only a limited number encoding a second aspartyl-tRNA synthetase.

Relaxed tRNA specificity of the Staphylococcus aureus aspartyl-tRNA synthetase enables RNA-dependent asparagine biosynthesis.

The human pathogen Staphylococcus aureus is an asparagine prototroph despite its genome not encoding an asparagine synthetase. S. aureus does use an asparaginyl-tRNA synthetase (AsnRS) to directly ligate asparagine to tRNA(Asn). The S. aureus genome also codes for one aspartyl-tRNA synthetase (AspRS). Here we demonstrate the lone S. aureus aspartyl-tRNA synthetase has relaxed tRNA specificity and can be used with the amidotransferase GatCAB to synthesize asparagine on tRNA(Asn). S. aureus thus encodes both the direct and indirect routes for Asn-tRNA(Asn) formation while encoding only one aspartyl-tRNA synthetase. The presence of the indirect pathway explains how S. aureus synthesizes asparagine without either asparagine synthetase.

Tracking molecular recognition at the atomic level with a new protein scaffold based on the OB-fold.

The OB-fold is a small, versatile single-domain protein binding module that occurs in all forms of life, where it binds protein, carbohydrate, nucleic acid and small-molecule ligands. We have exploited this natural plasticity to engineer a new class of non-immunoglobulin alternatives to antibodies with unique structural and biophysical characteristics. We present here the engineering of the OB-fold anticodon recognition domain from aspartyl tRNA synthetase taken from the thermophile Pyrobaculum aerophilum. For this single-domain scaffold we have coined the term OBody. Starting from a naïve combinatorial library, we engineered an OBody with 3 nM affinity for hen egg-white lysozyme, by optimising the affinity of a naïve OBody 11,700-fold over several affinity maturation steps, using phage display. At each maturation step a crystal structure of the engineered OBody in complex with hen egg-white lysozyme was determined, showing binding elements in atomic detail. These structures have given us an unprecedented insight into the directed evolution of affinity for a single antigen on the molecular scale. The engineered OBodies retain the high thermal stability of the parental OB-fold despite mutation of up to 22% of their residues. They can be expressed in soluble form and also purified from bacteria at high yields. They also lack disulfide bonds. These data demonstrate the potential of OBodies as a new scaffold for the engineering of specific binding reagents and provide a platform for further development of future OBody-based applications.

Released selective pressure on a structural domain gives new insights on the functional relaxation of mitochondrial aspartyl-tRNA synthetase.

Mammalian mitochondrial aminoacyl-tRNA synthetases are nuclear-encoded enzymes that are essential for mitochondrial protein synthesis. Due to an endosymbiotic origin of the mitochondria, many of them share structural domains with homologous bacterial enzymes of same specificity. This is also the case for human mitochondrial aspartyl-tRNA synthetase (AspRS) that shares the so-called bacterial insertion domain with bacterial homologs. The function of this domain in the mitochondrial proteins is unclear. Here, we show by bioinformatic analyses that the sequences coding for the bacterial insertion domain are less conserved in opisthokont and protist than in bacteria and viridiplantae. The divergence suggests a loss of evolutionary pressure on this domain for non-plant mitochondrial AspRSs. This discovery is further connected with the herein described occurrence of alternatively spliced transcripts of the mRNAs coding for some mammalian mitochondrial AspRSs. Interestingly, the spliced transcripts alternately lack one of the four exons that code for the bacterial insertion domain. Although we showed that the human alternative transcript is present in all tested tissues; co-exists with the full-length form, possesses 5'- and 3'-UTRs, a poly-A tail and is bound to polysomes, we were unable to detect the corresponding protein. The relaxed selective pressure combined with the occurrence of alternative splicing, involving a single structural sub-domain, favors the hypothesis of the loss of function of this domain for AspRSs of mitochondrial location. This evolutionary divergence is in line with other characteristics, established for the human mt-AspRS, that indicate a functional relaxation of non-viridiplantae mt-AspRSs when compared to bacterial and plant ones, despite their common ancestry.

Molecular evolution of protein-RNA mimicry as a mechanism for translational control.

Elongation factor P (EF-P) is a conserved ribosome-binding protein that structurally mimics tRNA to enable the synthesis of peptides containing motifs that otherwise would induce translational stalling, including polyproline. In many bacteria, EF-P function requires post-translational modification with (R)-ß-lysine by the lysyl-tRNA synthetase paralog PoxA. To investigate how recognition of EF-P by PoxA evolved from tRNA recognition by aminoacyl-tRNA synthetases, we compared the roles of EF-P/PoxA polar contacts with analogous interactions in a closely related tRNA/synthetase complex. PoxA was found to recognize EF-P solely via identity elements in the acceptor loop, the domain of the protein that interacts with the ribosome peptidyl transferase center and mimics the 3'-acceptor stem of tRNA. Although the EF-P acceptor loop residues required for PoxA recognition are highly conserved, their conservation was found to be independent of the phylogenetic distribution of PoxA. This suggests EF-P first evolved tRNA mimicry to optimize interactions with the ribosome, with PoxA-catalyzed aminoacylation evolving later as a secondary mechanism to further improve ribosome binding and translation control.