Probing electrostatic interactions and ligand binding in aspartyl-tRNA synthetase through site-directed mutagenesis and computer simulations

Faithful genetic code translation requires that each aminoacyl-tRNA synthetase recognise its cognate amino acid ligand specifically. Aspartyl-tRNA synthetase (AspRS) distinguishes between its negatively-charged Asp substrate and two competitors, neutral Asn and di-negative succinate, using a complex network of electrostatic interactions. Here, we used molecular dynamics simulations and site-directed mutagenesis experiments to probe these interactions further. We attempt to decrease the Asp/Asn binding free energy difference via single, double and triple mutations that reduce the net positive charge in the active site of Escherichia coli AspRS. Earlier, Glutamine 199 was changed to a negatively-charged glutamate, giving a computed reduction in Asp affinity in good agreement with experiment. Here, Lysine 198 was changed to a neutral leucine; then, Lys198 and Gln199 were mutated simultaneously. Both mutants are predicted to have reduced Asp binding and improved Asn binding, but the changes are insufficient to overcome the initial, high specificity of the native enzyme, which retains a preference for Asp. Probing the aminoacyl-adenylation reaction through pyrophosphate exchange experiments, we found no detectable activity for the mutant enzymes, indicating weaker Asp binding and/or poorer transition state stabilization. The simulations show that the mutations' effect is partly offset by proton uptake by a nearby histidine. Therefore, we performed additional simulations where the nearby Histidines 448 and 449 were mutated to neutral or negative residues: (Lys198Leu, His448Gln, His449Gln), and (Lys198Leu, His448Glu, His449Gln). This led to unexpected conformational changes and loss of active site preorganization, suggesting that the AspRS active site has a limited structural tolerance for electrostatic modifications. The data give insights into the complex electrostatic network in the AspRS active site and illustrate the difficulty in engineering charged-to-neutral changes of the preferred ligand.     

Molecular dynamics simulations show that bound Mg2+ contributes to amino acid and aminoacyl adenylate binding specificity in aspartyl-tRNA synthetase through long range electrostatic interactions

Molecular recognition between the aminoacyl-tRNA synthetase enzymes and their cognate amino acid ligands is essential for the faithful translation of the genetic code. In aspartyl-tRNA synthetase (AspRS), the co-substrate ATP binds preferentially with three associated Mg2+ cations in an unusual, bent geometry. The Mg2+ cations play a structural role and are thought to also participate catalytically in the enzyme reaction. Co-binding of the ATP x Mg3(2+) complex was shown recently to increase the Asp/Asn binding free energy difference, indicating that amino acid discrimination is substrate-assisted. Here, we used molecular dynamics free energy simulations and continuum electrostatic calculations to resolve two related questions. First, we showed that if one of the Mg2+ cations is removed, the Asp/Asn binding specificity is strongly reduced. Second, we computed the relative stabilities of the three-cation complex and the 2-cation complexes. We found that the 3-cation complex is overwhelmingly favored at ordinary magnesium concentrations, so that the protein is protected against the 2-cation state. In the homologous LysRS, the 3-cation complex was also strongly favored, but the third cation did not affect Lys binding. In tRNA-bound AspRS, the single remaining Mg2+ cation strongly favored the Asp-adenylate substrate relative to Asn-adenylate. Thus, in addition to their structural and catalytic roles, the Mg2+ cations contribute to specificity in AspRS through long range electrostatic interactions with the Asp side chain in both the pre- and post-adenylation states.     

The free yeast aspartyl-tRNA synthetase differs from the tRNA(Asp)-complexed enzyme by structural changes in the catalytic site, hinge region, and anticodon-binding domain

Aminoacyl-tRNA synthetases catalyze the specific charging of amino acid residues on tRNAs. Accurate recognition of a tRNA by its synthetase is achieved through sequence and structural signalling. It has been shown that tRNAs undergo large conformational changes upon binding to enzymes, but little is known about the conformational rearrangements in tRNA-bound synthetases. To address this issue the crystal structure of the dimeric class II aspartyl-tRNA synthetase (AspRS) from yeast was solved in its free form and compared to that of the protein associated to the cognate tRNA(Asp). The use of an enzyme truncated in N terminus improved the crystal quality and allowed us to solve and refine the structure of free AspRS at 2.3 A resolution. For the first time, snapshots are available for the different macromolecular states belonging to the same tRNA aminoacylation system, comprising the free forms for tRNA and enzyme, and their complex. Overall, the synthetase is less affected by the association than the tRNA, although significant local changes occur. They concern a rotation of the anticodon binding domain and a movement in the hinge region which connects the anticodon binding and active-site domains in the AspRS subunit. The most dramatic differences are observed in two evolutionary conserved loops. Both are in the neighborhood of the catalytic site and are of importance for ligand binding. The combination of this structural analysis with mutagenesis and enzymology data points to a tRNA binding process that starts by a recognition event between the tRNA anticodon loop and the synthetase anticodon binding module.     

An intricate RNA structure with two tRNA-derived motifs directs complex formation between yeast aspartyl-tRNA synthetase and its mRNA

Accurate translation of genetic information necessitates the tuned expression of a large group of genes. Amongst them, controlled expression of the enzymes catalyzing the aminoacylation of tRNAs, the aminoacyl-tRNA synthetases, is essential to insure translational fidelity. In the yeast Saccharomyces cerevisiae, expression of aspartyl-tRNA synthetase (AspRS) is regulated in a process necessitating recognition of the 5' extremity of AspRS messenger RNA (mRNA(AspRS)) by its translation product and adaptation to the cellular tRNA(Asp) concentration. Here, we have established the folding of the approximately 300 nucleotides long 5' end of mRNA(AspRS) and identified the structural signals involved in the regulation process. We show that the regulatory region in mRNA(AspRS) folds in two independent and symmetrically structured domains spaced by two single-stranded connectors. Domain I displays a tRNA(Asp) anticodon-like stem-loop structure with mimics of the aspartate identity determinants, that is restricted in domain II to a short double-stranded helix. The overall mRNA structure, based on enzymatic and chemical probing, supports a three-dimensional model where each monomer of yeast AspRS binds one individual domain and recognizes the mRNA structure as it recognizes its cognate tRNA(Asp). Sequence comparison of yeast genomes shows that the features within the mRNA recognized by AspRS are conserved in different Saccharomyces species. In the recognition process, the N-terminal extension of each AspRS subunit plays a crucial role in anchoring the tRNA-like motifs of the mRNA on the synthetase.     

The nondiscriminating aspartyl-tRNA synthetase from Helicobacter pylori: anticodon-binding domain mutations that impact tRNA specificity and heterologous toxicity

Divergent tRNA substrate recognition patterns distinguish the two distinct forms of aspartyl-tRNA synthetase (AspRS) that exist in different bacteria. In some cases, a canonical, discriminating AspRS (D-AspRS) specifically generates Asp-tRNA(Asp) and usually coexists with asparaginyl-tRNA synthetase (AsnRS). In other bacteria, particularly those that lack AsnRS, AspRS is nondiscriminating (ND-AspRS) and generates both Asp-tRNA(Asp) and the noncanonical, misacylated Asp-tRNA(Asn); this misacylated tRNA is subsequently repaired by the glutamine-dependent Asp-tRNA(Asn)/Glu-tRNA(Gln) amidotransferase (Asp/Glu-Adt). The molecular features that distinguish the closely related bacterial D-AspRS and ND-AspRS are not well-understood. Here, we report the first characterization of the ND-AspRS from the human pathogen Helicobacter pylori (H. pylori or Hp). This enzyme is toxic when heterologously overexpressed in Escherichia coli. This toxicity is rescued upon coexpression of the Hp Asp/Glu-Adt, indicating that Hp Asp/Glu-Adt can utilize E. coli Asp-tRNA(Asn) as a substrate. Finally, mutations in the anticodon-binding domain of Hp ND-AspRS reduce this enzyme's ability to misacylate tRNA(Asn), in a manner that correlates with the toxicity of the enzyme in E. coli.     

Thermus thermophilus contains an eubacterial and an archaebacterial aspartyl-tRNA synthetase

Thermus thermophilus possesses two aspartyl-tRNA synthetases (AspRSs), AspRS1 and AspRS2, encoded by distinct genes. Alignment of the protein sequences with AspRSs of other origins reveals that AspRS1 possesses the structural features of eubacterial AspRSs, whereas AspRS2 is structurally related to the archaebacterial AspRSs. The structural dissimilarity between the two thermophilic AspRSs is correlated with functional divergences. AspRS1 aspartylates tRNA(Asp) whereas AspRS2 aspartylates tRNA(Asp), and tRNA(Asn) with similar efficiencies. Since Asp bound on tRNA(Asn) is converted into Asn by a tRNA-dependent aspartate amidotransferase, AspRS2 is involved in Asn-tRNA(Asn) formation. These properties relate functionally AspRS2 to archaebacterial AspRSs. The structural basis of the dual specificity of T. thermophilus tRNA(Asn) was investigated by comparing its sequence with those of tRNA(Asp) and tRNA(Asn) of strict specificity. It is shown that the thermophilic tRNA(Asn) contains the elements defining asparagine identity in Escherichia coli, part of which being also the major elements of aspartate identity, whereas minor elements of this identity are missing. The structural context that permits expression of aspartate and asparagine identities by tRNA(Asn) and how AspRS2 accommodates tRNA(Asp) and tRNA(Asn) will be discussed. This work establishes a distinct structure-function relationship of eubacterial and archaebacterial AspRSs. The structural and functional properties of the two thermophilic AspRSs will be discussed in the context of the modern and primitive pathways of tRNA aspartylation and asparaginylation and related to the phylogenetic connexion of T. thermophilus to eubacteria and archaebacteria.     

A domain in the N-terminal extension of class IIb eukaryotic aminoacyl-tRNA synthetases is important for tRNA binding

Cytoplasmic aspartyl-tRNA synthetase (AspRS) from Saccharomyces cerevisiae is a homodimer of 64 kDa subunits. Previous studies have emphasized the high sensitivity of the N-terminal region to proteolytic cleavage, leading to truncated species that have lost the first 20-70 residues but that retain enzymatic activity and dimeric structure. In this work, we demonstrate that the N-terminal extension in yeast AspRS participates in tRNA binding and we generalize this finding to eukaryotic class IIb aminoacyl-tRNA synthetases. By gel retardation studies and footprinting experiments on yeast tRNA(Asp), we show that the extension, connected to the anticodon-binding module of the synthetase, contacts tRNA on the minor groove side of its anticodon stem. Sequence comparison of eukaryotic class IIb synthetases identifies a lysine-rich 11 residue sequence ((29)LSKKALKKLQK(39) in yeast AspRS with the consensus xSKxxLKKxxK in class IIb synthetases) that is important for this binding. Direct proof of the role of this sequence comes from a mutagenesis analysis and from binding studies using the isolated peptide.     

Non-essential role of lysine residues for the catalytic activities of aspartyl-tRNA synthetase and comparison with other aminoacyl-tRNA synthetases

Essential lysine residues were sought in the catalytic site of baker's yeast aspartyl-tRNA synthetase (an alpha 2 dimer of Mr 125,000) using affinity labeling methods and periodate-oxidized adenosine, ATP, and tRNA(Asp). It is shown that the number of periodate-oxidized derivatives which can be bound to the synthetase via Schiff's base formation with epsilon-NH2 groups of lysine residues exceeds the stoichiometry of specific substrate binding. Furthermore, it is found that the enzymatic activities are not completely abolished, even for high incorporation levels of the modified substrates. The tRNA(Asp) aminoacylation reaction is more sensitive to labeling than is the ATP-PPi exchange one; for enzyme preparations modified with oxidized adenosine or ATP this activity remains unaltered. These results demonstrate the absence of a specific lysine residue directly involved in the catalytic activities of yeast aspartyl-tRNA synthetase. Comparative labeling experiments with oxidized ATP were run with several other aminoacyl-tRNA synthetases. Residual ATP-PPi exchange and tRNA aminoacylation activities measured in each case on the modified synthetases reveal different behaviors of these enzymes when compared to that of aspartyl-tRNA synthetase. When tested under identical experimental conditions, pure isoleucyl-, methionyl-, threonyl- and valyl-tRNA synthetases from E. coli can be completely inactivated for their catalytic activities; for E. coli alanyl-tRNA synthetase only the tRNA charging activity is affected, whereas yeast valyl-tRNA synthetase is only partly inactivated. The structural significance of these experiments and the occurrence of essential lysine residues in aminoacyl-tRNA synthetases are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ammonium scanning in an enzyme active site. The chiral specificity of aspartyl-tRNA synthetase

D-amino acids are largely excluded from protein synthesis, yet they are of great interest in biotechnology. Aspartyl-tRNA synthetase (AspRS) can misacylate tRNA(Asp) with D-aspartate instead of its usual substrate, L-Asp. We investigate how the preference for L-Asp arises, using molecular dynamics simulations. Asp presents a special problem, having pseudosymmetry broken only by its ammonium group, and AspRS must protect not only against D-Asp, but against an "inverted" orientation where the two substrate carboxylates are swapped. We compare L-Asp and D-Asp, in both orientations, and succinate, where the ammonium group is removed and the ligand has an additional negative charge. All possible ammonium positions on the ligand are thus scanned, providing information on electrostatic interactions. As controls, we simulate a Q199E mutation, obtaining a reduction in binding free energy in agreement with experiment, and we simulate TyrRS, which can misacylate tRNA(Tyr) with D-Tyr. For both TyrRS and AspRS, we obtain a moderate binding free energy difference DeltaDeltaG between the L- and D-amino acids, in agreement with their known ability to misacylate their tRNAs. In contrast, we predict that AspRS is strongly protected against inverted L-Asp binding. For succinate, kinetic measurements reveal a DeltaDeltaG of over 5 kcal/mol, favoring L-Asp. The simulations show how chiral discriminations arises from the structures, with two AspRS conformations acting in different ways and proton uptake by nearby histidines playing a role. A complex network of charges protects AspRS against most binding errors, making the engineering of its specificity a difficult challenge.     

Non-discriminating and discriminating aspartyl-tRNA synthetases differ in the anticodon-binding domain

In most organisms, tRNA aminoacylation is ensured by 20 aminoacyl-tRNA synthetases (aaRSs). In eubacteria, however, synthetases can be duplicated as in Thermus thermophilus, which contains two distinct AspRSs. While AspRS-1 is specific, AspRS-2 is non-discriminating and aspartylates tRNA(Asp) and tRNA(Asn). The structure at 2.3 A resolution of AspRS-2, the first of a non-discriminating synthetase, was solved. It differs from that of AspRS-1 but has resemblance to that of discriminating and archaeal AspRS from Pyrococcus kodakaraensis. The protein presents non-conventional features in its OB-fold anticodon-binding domain, namely the absence of a helix inserted between two beta-strands of this fold and a peculiar L1 loop differing from the large loops known to interact with tRNA(Asp) identity determinant C36 in conventional AspRSs. In AspRS-2, this loop is small and structurally homologous to that in AsnRSs, including conservation of a proline. In discriminating Pyrococcus AspRS, the L1 loop, although small, lacks this proline and is not superimposable with that of AspRS-2 or AsnRS. Its particular status is demonstrated by a loop-exchange experiment that renders the Pyrococcus AspRS non-discriminating.     

cDNA sequence, predicted primary structure, and evolving amphiphilic helix of human aspartyl-tRNA synthetase

Eight of the mammalian aminoacyl-tRNA synthetases associate as a multienzyme complex, whereas prokaryotic and low eukaryotic synthetases occur only as free soluble enzymes. Association of the synthetases may result in effective compartmentalization of synthetases and suggests the association of the entire protein biosynthetic machinery. To elucidate the structural elements and the nature of the molecular interactions involved in the association of the synthetases, we have cloned and sequenced the complementary DNA coding human aspartyl-tRNA synthetase. The full length cDNA encodes an open reading frame of 500 amino acids with 56% identity with yeast aspartyl-tRNA synthetase. The similarity with yeast aspartyl-tRNA synthetase is unevenly distributed with a high percent of identity at the C-terminus and relatively low identity at the N-terminus. The N-terminal sequence strongly prefers an alpha-helical secondary structure and shows amphiphilic characteristics. Further comparison with the yeast synthetases showed that the basic positively charged helixes in yeast synthetases are evolved to a neutral amphiphilic helix in this mammalian synthetase. The mammalian neutral amphiphilic helix is so far unique among all known sequences of bacterial, yeast, and mammalian synthetases and may account for the association of synthetases in the synthetase complex.     

Two residues in the anticodon recognition domain of the aspartyl-tRNA synthetase from Pseudomonas aeruginosa are individually implicated in the recognition of tRNAAsn

In many organisms, the formation of asparaginyl-tRNA is not done by direct aminoacylation of tRNA(Asn) but by specific tRNA-dependent transamidation of aspartyl-tRNA(Asn). This transamidation pathway involves a nondiscriminating aspartyl-tRNA synthetase (AspRS) that charges both tRNA(Asp) and tRNA(Asn) with aspartic acid. Recently, it has been shown for the first time in an organism (Pseudomonas aeruginosa PAO1) that the transamidation pathway is the only route of synthesis of Asn-tRNA(Asn) but does not participate in Gln-tRNA(Gln) formation. P. aeruginosa PAO1 has a nondiscriminating AspRS. We report here the identification of two residues in the anticodon recognition domain (H31 and G83) which are implicated in the recognition of tRNA(Asn). Sequence comparisons of putative discriminating and nondiscriminating AspRSs (based on the presence or absence of the AdT operon and of AsnRS) revealed that bacterial nondiscriminating AspRSs possess a histidine at position 31 and usually a glycine at position 83, whereas discriminating AspRSs possess a leucine at position 31 and a residue other than a glycine at position 83. Mutagenesis of these residues of P. aeruginosa AspRS from histidine to leucine and from glycine to lysine increased the specificity of tRNA(Asp) charging over that of tRNA(Asn) by 3.5-fold and 4.2-fold, respectively. Thus, we show these residues to be determinants of the relaxed specificity of this nondiscriminating AspRS. Using available crystallographic data, we found that the H31 residue could interact with the central bases of the anticodons of the tRNA(Asp) and tRNA(Asn). Therefore, these two determinants of specificity of P. aeruginosa AspRS could be important for all bacterial AspRSs.     

A single amidotransferase forms asparaginyl-tRNA and glutaminyl-tRNA in Chlamydia trachomatis

Aminoacyl-tRNA is generally formed by aminoacyl-tRNA synthetases, a family of 20 enzymes essential for accurate protein synthesis. However, most bacteria generate one of the two amide aminoacyl-tRNAs, Asn-tRNA or Gln-tRNA, by transamidation of mischarged Asp-tRNA(Asn) or Glu-tRNA(Gln) catalyzed by a heterotrimeric amidotransferase (encoded by the gatA, gatB, and gatC genes). The Chlamydia trachomatis genome sequence reveals genes for 18 synthetases, whereas those for asparaginyl-tRNA synthetase and glutaminyl-tRNA synthetase are absent. Yet the genome harbors three gat genes in an operon-like arrangement (gatCAB). We reasoned that Chlamydia uses the gatCAB-encoded amidotransferase to generate both Asn-tRNA and Gln-tRNA. C. trachomatis aspartyl-tRNA synthetase and glutamyl-tRNA synthetase were shown to be non-discriminating synthetases that form the misacylated tRNA(Asn) and tRNA(Gln) species. A preparation of pure heterotrimeric recombinant C. trachomatis amidotransferase converted Asp-tRNA(Asn) and Glu-tRNA(Gln) into Asn-tRNA and Gln-tRNA, respectively. The enzyme used glutamine, asparagine, or ammonia as amide donors in the presence of either ATP or GTP. These results suggest that C. trachomatis employs the dual specificity gatCAB-encoded amidotransferase and 18 aminoacyl-tRNA synthetases to create the complete set of 20 aminoacyl-tRNAs.     

Aspartyl-tRNA synthetase requires a conserved proline in the anticodon-binding loop for tRNA(Asn) recognition in vivo

Most prokaryotes require Asp-tRNA(Asn) for the synthesis of Asn-tRNA(Asn). This misacylated tRNA species is synthesized by a non-discriminating aspartyl-tRNA synthetase (AspRS) that acylates both tRNA(Asp) and tRNA(Asn) with aspartate. In contrast, a discriminating AspRS forms only Asp-tRNA(Asp). Here we show that a conserved proline (position 77) in the L1 loop of the non-discriminating Deinococcus radiodurans AspRS2 is required for tRNA(Asn) recognition in vivo. Escherichia coli trpA34 was transformed with DNA from a library of D. radiodurans aspS2 genes with a randomized codon 77 and then subjected to in vivo selection for Asp-tRNA(Asn) formation by growth in minimal medium. Only proline codons were found at position 77 in the aspS2 genes isolated from 21 of the resulting viable colonies. However, when the aspS temperature-sensitive E. coli strain CS89 was transformed with the same DNA library and then screened for Asp-tRNA(Asp) formation in vivo by growth at the non-permissive temperature, codons for seven other amino acids besides proline were identified at position 77 in the isolates examined. Thus, replacement of proline 77 by cysteine, isoleucine, leucine, lysine, phenylalanine, serine, or valine resulted in mutant D. radiodurans AspRS2 enzymes still capable of forming Asp-tRNA(Asp) but unable to recognize tRNA(Asn). This strongly suggests that proline 77 is responsible for the non-discriminatory tRNA recognition properties of this enzyme.     

Yeast aspartyl-tRNA synthetase binds specifically its own mRNA

Dimeric class II aspartyl-tRNA synthetase (AspRS) from yeast has a modular architecture and includes an N-terminal appendix of 70 amino acid residues that protrudes from the anticodon-binding module. This extension, of predicted helical structure, is not essential for aminoacylation but contains an RNA-binding motif that promotes non-specific interactions with tRNAs. As shown here, this protein extension can also interact with the 5' end of the AspRS mRNA. In vitro, optimal binding occurs on an mRNA domain comprising part of the 87 nucleotide long 5'UTR and the sequence encoding the N-terminal appendix. At the protein side, only the appendix and the anticodon-binding module participate in the interaction between AspRS and the mRNA domain. Binding is specific, since only tRNA(Asp) can dissociate the complex. In vivo, AspRS also binds specifically this mRNA domain and in doing so triggers a reduced translation of a fused GFP mRNA. From that, a mechanism for the regulation of this eukaryotic aminoacyl-tRNA synthetase is proposed. Implications for aspartylation accuracy in yeast are given.     

tRNA-balanced expression of a eukaryal aminoacyl-tRNA synthetase by an mRNA-mediated pathway

Aminoacylation of transfer RNAs is a key step during translation. It is catalysed by the aminoacyl-tRNA synthetases (aaRSs) and requires the specific recognition of their cognate substrates, one or several tRNAs, ATP and the amino acid. Whereas the control of certain aaRS genes is well known in prokaryotes, little is known about the regulation of eukaryotic aaRS genes. Here, it is shown that expression of AspRS is regulated in yeast by a feedback mechanism that necessitates the binding of AspRS to its messenger RNA. This regulation leads to a synchronized expression of AspRS and tRNA(Asp). The correlation between AspRS expression and mRNA(AspRS) and tRNA(Asp) concentrations, as well as the presence of AspRS in the nucleus, suggests an original regulation mechanism. It is proposed that the surplus of AspRS, not sequestered by tRNA(Asp), is imported into the nucleus where it binds to mRNA(AspRS) and thus inhibits its accumulation.