Crystal structure of glutamyl-queuosine tRNAAsp synthetase complexed with L-glutamate: structural elements mediating tRNA-independent activation of glutamate and glutamylation of tRNAAsp anticodon

Glutamyl-queuosine tRNA^Asp synthetase (Glu-Q-RS) from Escherichia coli is a paralog of the catalytic core of glutamyl-tRNA synthetase (GluRS) that catalyzes glutamylation of queuosine in the wobble position of tRNA^Asp. Despite important structural similarities, Glu-Q-RS and GluRS diverge strongly by their functional properties. The only feature common to both enzymes consists in the activation of Glu to form Glu-AMP, the intermediate of transfer RNA (tRNA) aminoacylation. However, both enzymes differ by the mechanism of selection of the cognate amino acid and by the mechanism of its activation. Whereas GluRS selects l-Glu and activates it only in the presence of the cognate tRNA^Glu, Glu-Q-RS forms Glu-AMP in the absence of tRNA. Moreover, while GluRS transfers the activated Glu to the 3′ accepting end of the cognate tRNA^Glu, Glu-Q-RS transfers the activated Glu to Q34 located in the anticodon loop of the noncognate tRNA^Asp. In order to gain insight into the structural elements leading to distinct mechanisms of amino acid activation, we solved the three-dimensional structure of Glu-Q-RS complexed to Glu and compared it to the structure of the GluRS·Glu complex. Comparison of the catalytic site of Glu-Q-RS with that of GluRS, combined with binding experiments of amino acids, shows that a restricted number of residues determine distinct catalytic properties of amino acid recognition and activation by the two enzymes. Furthermore, to explore the structural basis of the distinct aminoacylation properties of the two enzymes and to understand why Glu-Q-RS glutamylates only tRNA^Asp among the tRNAs possessing queuosine in position 34, we performed a tRNA mutational analysis to search for the elements of tRNA^Asp that determine recognition by Glu-Q-RS. The analyses made on tRNA^Asp and tRNA^Asn show that the presence of a C in position 38 is crucial for glutamylation of Q34. The results are discussed in the context of the evolution and adaptation of the tRNA glutamylation system.     

Dispensability of zinc and the putative zinc-binding domain in bacterial glutamyl-tRNA synthetase

The putative zinc-binding domain (pZBD) in Escherichia coli glutamyl-tRNA synthetase (GluRS) is known to correctly position the tRNA acceptor arm and modulate the amino acid-binding site. However, its functional role in other bacterial species is not clear since many bacterial GluRSs lack a zinc-binding motif in the pZBD. From experimental studies on pZBD-swapped E. coli GluRS, with Thermosynechoccus elongatus GluRS, Burkholderia thailandensis GluRS and E. coli glutamyl-queuosine-tRNA^Asp synthetase (Glu-Q-RS), we show that E. coli GluRS, containing the zinc-free pZBD of B. thailandensis, is as functional as the zinc-bound wild-type E. coli GluRS, whereas the other constructs, all zinc-bound, show impaired function. A pZBD-tinkered version of E. coli GluRS that still retained Zn-binding capacity, also showed reduced activity. This suggests that zinc is not essential for the pZBD to be functional. From extensive structural and sequence analyses from whole genome database of bacterial GluRS, we further show that in addition to many bacterial GluRS lacking a zinc-binding motif, the pZBD is actually deleted in some bacteria, all containing either glutaminyl-tRNA synthetase (GlnRS) or a second copy of GluRS (GluRS2). Correlation between the absence of pZBD and the occurrence of glutamine amidotransferase CAB (GatCAB) in the genome suggests that the primordial role of the pZBD was to facilitate transamidation of misacylated Glu-tRNA^Gln via interaction with GatCAB, whereas its role in tRNA^Glu interaction may be a consequence of the presence of pZBD.     

Fusion with Anticodon Binding Domain of GluRS is Not Sufficient to Alter the Substrate Specificity of a Chimeric Glu-Q-RS

Glutamyl-queuosine-tRNA^Asp synthetase (Glu-Q-RS) is a paralog of glutamyl-tRNA synthetase (GluRS) and is found in more than forty species of proteobacteria, cyanobacteria, and actinobacteria. Glu-Q-RS shows striking structural similarity with N-terminal catalytic domain of GluRS (NGluRS) but it lacks the C-terminal anticodon binding domain (CGluRS). In spite of structural similarities, Glu-Q-RS and NGluRS differ in their functional properties. Glu-Q-RS glutamylates the Q34 nucleotide of the anticodon of tRNA^Asp whereas NGluRS constitutes the catalytic domain of GluRS catalyzing the transfer of Glu on the acceptor end of tRNA^Glu. Since NGluRS is able to catalyze aminoacylation of only tRNA^Glu the glutamylation capacity of tRNA^Asp by Glu-Q-RS is surprising. To understand the substrate specificity of Glu-Q-RS we undertook a systemic approach by investigating the biophysical and biochemical properties of the NGluRS (1–301), CGluRS (314–471) and Glu-Q-RS-CGluRS, (1–298 of Glu-Q-RS fused to 314–471 from GluRS). Circular dichroism, fluorescence spectroscopy and differential scanning calorimetry analyses revealed absence of N-terminal domain (1–298 of Glu-Q-RS) and C-terminal domain (314–471 from GluRS) communication in chimera, in contrast to the native full length GluRS. The chimeric Glu-Q-RS is still able to aminoacylate tRNA^Asp but has also the capacity to bind tRNA^Glu. However the chimeric protein is unable to aminoacylate tRNA^Glu probably as a consequence of the lack of domain–domain communication.     

The role of the catalytic domain of E. coli GluRS in tRNA discrimination

Discrimination of tRNA^Gln is an integral function of several bacterial glutamyl-tRNA synthetases (GluRS). The origin of the discrimination is thought to arise from unfavorable interactions between tRNA^Gln and the anticodon-binding domain of GluRS. From experiments on an anticodon-binding domain truncated Escherichia coli (E. coli) GluRS (catalytic domain) and a chimeric protein, constructed from the catalytic domain of E. coli GluRS and the anticodon-binding domain of E. coli glutaminyl-tRNA synthetase (GlnRS), we show that both proteins discriminate against E. coli tRNA^Gln. Our results demonstrate that in addition to the anticodon-binding domain, tRNA^Gln discriminatory elements may be present in the catalytic domain in E. coli GluRS as well.     

The zinc-binding site of a class I aminoacyl-tRNA synthetase is a SWIM domain that modulates amino acid binding via the tRNA acceptor arm.

In its tRNA acceptor end binding domain, the glutamyl-tRNA synthetase (GluRS) of Escherichia coli contains one atom of zinc that holds the extremities of a segment (Cys98-x-Cys100-x24-Cys125-x-His127) homologous to the Escherichia coli glutaminyl-tRNA synthetase (GlnRS) loop where a leucine residue stabilizes the peeled-back conformation of tRNAGln acceptor end. We report here that the GluRS zinc-binding region belongs to the novel SWIM domain family characterized by the signature C-x-C-xn-C-x-H (n = 6-25), and predicted to interact with DNA or proteins. In the presence of tRNAGlu, the GluRS C100Y variant has a lower affinity for l-glutamate than the wild-type enzyme, with Km and Kd values increased 12- and 20-fold, respectively. On the other hand, in the absence of tRNAGlu, glutamate binds with the same affinity to the C100Y variant and to wild-type GluRS. In the context of the close structural and mechanistic similarities between GluRS and GlnRS, these results indicate that the GluRS SWIM domain modulates glutamate binding to the active site via its interaction with the tRNAGlu acceptor arm. Phylogenetic analyses indicate that ancestral GluRSs had a strong zinc-binding site in their SWIM domain. Considering that all GluRSs require a cognate tRNA to activate glutamate, and that some of them have different or no putative zinc-binding residues in the corresponding positions, the properties of the C100Y variant suggest that the GluRS SWIM domains evolved to position correctly the tRNA acceptor end in the active site, thereby contributing to the formation of the glutamate binding site.     

Coevolution of Specificity Determinants in Eukaryotic Glutamyl- and Glutaminyl-tRNA Synthetases

The glutaminyl-tRNA synthetase (GlnRS) enzyme, which pairs glutamine with tRNA^Gln for protein synthesis, evolved by gene duplication in early eukaryotes from a nondiscriminating glutamyl-tRNA synthetase (GluRS) that aminoacylates both tRNA^Gln and tRNA^Glu with glutamate. This ancient GluRS also separately differentiated to exclude tRNA^Gln as a substrate, and the resulting discriminating GluRS and GlnRS further acquired additional protein domains assisting function in cis (the GlnRS N-terminal Yqey domain) or in trans (the Arc1p protein associating with GluRS). These added domains are absent in contemporary bacterial GlnRS and GluRS. Here, using Saccharomyces cerevisiae enzymes as models, we find that the eukaryote-specific protein domains substantially influence amino acid binding, tRNA binding and aminoacylation efficiency, but they play no role in either specific nucleotide readout or discrimination against noncognate tRNA. Eukaryotic tRNA^Gln and tRNA^Glu recognition determinants are found in equivalent positions and are mutually exclusive to a significant degree, with key nucleotides located adjacent to portions of the protein structure that differentiated during the evolution of archaeal nondiscriminating GluRS to GlnRS. These findings provide important corroboration for the evolutionary model and suggest that the added eukaryotic domains arose in response to distinctive selective pressures associated with the greater complexity of the eukaryotic translational apparatus. We also find that the affinity of GluRS for glutamate is significantly increased when Arc1p is not associated with the enzyme. This is consistent with the lower concentration of intracellular glutamate and the dissociation of the Arc1p:GluRS complex upon the diauxic shift to respiratory conditions.     

tRNAGlu Increases the Affinity of Glutamyl-tRNA Synthetase for Its Inhibitor Glutamyl-Sulfamoyl-Adenosine,an Analogue of the Aminoacylation Reaction Intermediate Glutamyl-AMP: Mechanistic and Evolutionary Implications

For tRNA-dependent protein biosynthesis, amino acids are first activated by aminoacyl-tRNA synthetases (aaRSs) yielding the reaction intermediates aminoacyl-AMP (aa-AMP). Stable analogues of aa-AMP, such as aminoacyl-sulfamoyl-adenosines, inhibit their cognate aaRSs. Glutamyl-sulfamoyl-adenosine (Glu-AMS) is the best known inhibitor of Escherichia coli glutamyl-tRNA synthetase (GluRS). Thermodynamic parameters of the interactions between Glu-AMS and E. coli GluRS were measured in the presence and in the absence of tRNA by isothermal titration microcalorimetry. A significant entropic contribution for the interactions between Glu-AMS and GluRS in the absence of tRNA or in the presence of the cognate tRNA^Glu or of the non-cognate tRNA^Phe is indicated by the negative values of –TΔS_b, and by the negative value of ΔC_p. On the other hand, the large negative enthalpy is the dominant contribution to ΔG_b in the absence of tRNA. The affinity of GluRS for Glu-AMS is not altered in the presence of the non-cognate tRNA^Phe, but the dissociation constant K _d is decreased 50-fold in the presence of tRNA^Glu; this result is consistent with molecular dynamics results indicating the presence of an H-bond between Glu-AMS and the 3’-OH oxygen of the 3’-terminal ribose of tRNA^Glu in the Glu-AMS•GluRS•tRNA^Glu complex. Glu-AMS being a very close structural analogue of Glu-AMP, its weak binding to free GluRS suggests that the unstable Glu-AMP reaction intermediate binds weakly to GluRS; these results could explain why all the known GluRSs evolved to activate glutamate only in the presence of tRNA^Glu, the coupling of glutamate activation to its transfer to tRNA preventing unproductive cleavage of ATP.     

A Nondiscriminating Glutamyl-tRNA Synthetase in the Plasmodium Apicoplast: THE FIRST ENZYME IN AN INDIRECT AMINOACYLATION PATHWAY*

The malaria parasite Plasmodium falciparum and related organisms possess a relict plastid known as the apicoplast. Apicoplast protein synthesis is a validated drug target in malaria because antibiotics that inhibit translation in prokaryotes also inhibit apicoplast protein synthesis and are sometimes used for malaria prophylaxis or treatment. We identified components of an indirect aminoacylation pathway for Gln-tRNA^Gln biosynthesis in Plasmodium that we hypothesized would be essential for apicoplast protein synthesis. Here, we report our characterization of the first enzyme in this pathway, the apicoplast glutamyl-tRNA synthetase (GluRS). We expressed the recombinant P. falciparum enzyme in Escherichia coli, showed that it is nondiscriminating because it glutamylates both apicoplast tRNA^Glu and tRNA^Gln, determined its kinetic parameters, and demonstrated its inhibition by a known bacterial GluRS inhibitor. We also localized the Plasmodium berghei ortholog to the apicoplast in blood stage parasites but could not delete the PbGluRS gene. These data show that Gln-tRNA^Gln biosynthesis in the Plasmodium apicoplast proceeds via an essential indirect aminoacylation pathway that is reminiscent of bacteria and plastids.     

Recognition of tRNAGln by Helicobacter pylori GluRS2—a tRNAGln-specific glutamyl-tRNA synthetase

Accurate aminoacylation of tRNAs by the aminoacyl-tRNA synthetases (aaRSs) plays a critical role in protein translation. However, some of the aaRSs are missing in many microorganisms. Helicobacter pylori does not have a glutaminyl-tRNA synthetase (GlnRS) but has two divergent glutamyl-tRNA synthetases: GluRS1 and GluRS2. Like a canonical GluRS, GluRS1 aminoacylates tRNA^Glu1 and tRNA^Glu2. In contrast, GluRS2 only misacylates tRNA^Gln to form Glu-tRNA^Gln. It is not clear how GluRS2 achieves specific recognition of tRNA^Gln while rejecting the two H. pylori tRNA^Glu isoacceptors. Here, we show that GluRS2 recognizes major identity elements clustered in the tRNA^Gln acceptor stem. Mutations in the tRNA anticodon or at the discriminator base had little to no impact on enzyme specificity and activity.     

Misacylation of tRNA with methionine in Saccharomyces cerevisiae

Accurate transfer RNA (tRNA) aminoacylation by aminoacyl-tRNA synthetases controls translational fidelity. Although tRNA synthetases are generally highly accurate, recent results show that the methionyl-tRNA synthetase (MetRS) is an exception. MetRS readily misacylates non-methionyl tRNAs at frequencies of up to 10% in mammalian cells; such mismethionylation may serve a beneficial role for cells to protect their own proteins against oxidative damage. The Escherichia coli MetRS mismethionylates two E. coli tRNA species in vitro, and these two tRNAs contain identity elements for mismethionylation. Here we investigate tRNA mismethionylation in Saccharomyces cerevisiae. tRNA mismethionylation occurs at a similar extent in vivo as in mammalian cells. Both cognate and mismethionylated tRNAs have similar turnover kinetics upon cycloheximide treatment. We identify specific arginine/lysine to methionine-substituted peptides in proteomic mass spectrometry, indicating that mismethionylated tRNAs are used in translation. The yeast MetRS is part of a complex containing the anchoring protein Arc1p and the glutamyl-tRNA synthetase (GluRS). The recombinant Arc1p–MetRS–GluRS complex binds and mismethionylates many tRNA species in vitro. Our results indicate that the yeast MetRS is responsible for extensive misacylation of non-methionyl tRNAs, and mismethionylation also occurs in this evolutionary branch.     

Structure of an archaeal non-discriminating glutamyl-tRNA synthetase: a missing link in the evolution of Gln-tRNAGln formation

The molecular basis of the genetic code relies on the specific ligation of amino acids to their cognate tRNA molecules. However, two pathways exist for the formation of Gln-tRNA^Gln. The evolutionarily older indirect route utilizes a non-discriminating glutamyl-tRNA synthetase (ND-GluRS) that can form both Glu-tRNA^Glu and Glu-tRNA^Gln. The Glu-tRNA^Gln is then converted to Gln-tRNA^Gln by an amidotransferase. Since the well-characterized bacterial ND-GluRS enzymes recognize tRNA^Glu and tRNA^Gln with an unrelated α-helical cage domain in contrast to the β-barrel anticodon-binding domain in archaeal and eukaryotic GluRSs, the mode of tRNA^Glu/tRNA^Gln discrimination in archaea and eukaryotes was unknown. Here, we present the crystal structure of the Methanothermobacter thermautotrophicus ND-GluRS, which is the evolutionary predecessor of both the glutaminyl-tRNA synthetase (GlnRS) and the eukaryotic discriminating GluRS. Comparison with the previously solved structure of the Escherichia coli GlnRS-tRNA^Gln complex reveals the structural determinants responsible for specific tRNA^Gln recognition by GlnRS compared to promiscuous recognition of both tRNAs by the ND-GluRS. The structure also shows the amino acid recognition pocket of GluRS is more variable than that found in GlnRS. Phylogenetic analysis is used to reconstruct the key events in the evolution from indirect to direct genetic encoding of glutamine.     

Mechanistic studies on Pyrobaculum calidifontis porphobilinogen synthase (5-aminolevulinic acid dehydratase)

Porphobilinogen synthase (PBG synthase) gene from Pyrobaculum calidifontis was cloned and expressed in E. coli. The recombinant enzyme was purified as an octamer and was found by mass spectrometry to have a subunit M_r of 37676.59 (calculated, 37676.3). The enzyme showed high thermal stability and retained almost all of its activity after incubation at 70 °C for 16 h in the presence of β-mercaptoethanol (β-ME) and zinc chloride. However, in the absence of the latter the enzyme was inactivated after 16 h although it regained full activity in the presence of β-ME and zinc chloride. The protein contained 4 mol of tightly bound zinc per octamer. Further, 4 mol of low affinity zinc could be incorporated following incubation with exogenous zinc salts. The enzyme was inactivated by incubation with levulinic acid followed by treatment with sodium borohydride. Tryptic digest of the modified enzyme and mass spectrometric analysis showed that Lys²⁵⁷ was the site of modification, which has previously been shown to be the site for the binding of 5-aminolevulinic acid giving rise to the propionate-half of porphobilinogen. P. calidifontis PBG synthase was inactivated by 5-chlorolevulinic acid and the residue modified was shown to be the central cysteine (Cys¹²⁷) of the zinc-binding cysteine-triad, comprising Cys^{125, 127, 135}. The present results in conjunction with earlier findings on zinc containing PBG synthases, are discussed which advocate that the catalytic role of zinc in the activation of the 5-aminolevulinic acid molecule forming the acetate-half of PBG is possible.     

A Possible Role for Metallic Ions in the Carbohydrate Cluster Recognition Displayed by a Lewis Y Specific Antibody

Background

Lewis Y (Le^y) is a blood group-related carbohydrate that is expressed at high surface densities on the majority of epithelial carcinomas and is a promising target for antibody-based immunotherapy. A humanized Le^y-specific antibody (hu3S193) has shown encouraging safety, pharmacokinetic and tumor-targeting properties in recently completed Phase I clinical trials.

Methodology/Principal Findings

We report the three-dimensional structures for both the free (unliganded) and bound (Le^y tetrasaccharide) hu3S193 Fab from the same crystal grown in the presence of divalent zinc ions. There is no evidence of significant conformational changes occurring in either the Le^y carbohydrate antigen or the hu3S193 binding site, which suggests a rigid fit binding mechanism. In the crystal, the hu3S193 Fab molecules are coordinated at their protein-protein interface by two zinc ions and in solution aggregation of Fab can be initiated by zinc, but not magnesium ions. Dynamic light scattering revealed that zinc ions could initiate a sharp transition from hu3S193 Fab monomers to large multimeric aggregates in solution.

Conclusions/Significance

Zinc ions can mediate interactions between hu3S193 Fab in crystals and in solution. Whether metallic ion mediated aggregation of antibody occurs in vivo is not known, but the present results suggest that similar clustering mechanisms could occur when hu3S193 binds to Le^y on cells, particularly given the high surface densities of antigen on the target tumor cells.     

Redox status affects the catalytic activity of glutamyl-tRNA synthetase

Glutamyl-tRNA synthetases (GluRS) provide Glu-tRNA for different processes including protein synthesis, glutamine transamidation and tetrapyrrole biosynthesis. Many organisms contain multiple GluRSs, but whether these duplications solely broaden tRNA specificity or also play additional roles in tetrapyrrole biosynthesis is not known. Previous studies have shown that GluRS1, one of two GluRSs from the extremophile Acidithiobacillus ferrooxidans, is inactivated when intracellular heme is elevated suggesting a specific role for GluRS1 in the regulation of tetrapyrrole biosynthesis. We now show that, in vitro, GluRS1 activity is reversibly inactivated upon oxidation by hemin and hydrogen peroxide. The targets for oxidation-based inhibition were found to be cysteines from a SWIM zinc-binding motif located in the tRNA acceptor helix-binding domain. tRNA^Glu was able to protect GluRS1 against oxidative inactivation by hemin plus hydrogen peroxide. The sensitivity to oxidation of A. ferrooxidans GluRS1 might provide a means to regulate tetrapyrrole and protein biosynthesis in response to extreme changes in both the redox and heme status of the cell via a single enzyme.     

Suppression of Amber Codons in Caulobacter crescentus by the Orthogonal Escherichia coli Histidyl-tRNA Synthetase/tRNAHis Pair

While translational read-through of stop codons by suppressor tRNAs is common in many bacteria, archaea and eukaryotes, this phenomenon has not yet been observed in the α-proteobacterium Caulobacter crescentus. Based on a previous report that C. crescentus and Escherichia coli tRNA^His have distinctive identity elements, we constructed E. coli tRNA^His _CUA, a UAG suppressor tRNA for C. crescentus. By examining the expression of three UAG codon- containing reporter genes (encoding a β-lactamase, the fluorescent mCherry protein, or the C. crescentus xylonate dehydratase), we demonstrated that the E. coli histidyl-tRNA synthetase/tRNA^His _CUA pair enables in vivo UAG suppression in C. crescentus. E. coli histidyl-tRNA synthetase (HisRS) or tRNA^His _CUA alone did not achieve suppression; this indicates that the E. coli HisRS/tRNA^His _CUA pair is orthogonal in C. crescentus. These results illustrate that UAG suppression can be achieved in C. crescentus with an orthogonal aminoacyl-tRNA synthetase/suppressor tRNA pair.     

Cloning and characterization of mitochondrial methionyl-tRNA synthetase from a pathogenic fungi Candida albicans

A genomic sequence encoding mitochondrial methionyl-tRNA synthetase (MetRS) was determined from a pathogenic fungi Candida albicans. The gene is distinct from that encoding the cytoplasmic MetRS. The encoded protein consists of 577 amino acids (aa) and contains the class I defining sequences in the N-terminal domain and the conserved anticodon-binding amino acid, Trp, in the C-terminal domain. This protein showed the highest similarity with the mitochondrial MetRSs of Saccharomyces cerevisiae and Shizosaccharomyces pombe. The mitochondrial MetRSs of these fungi were distinguished from their cytoplasmic forms. The protein lacks the zinc binding motif in the N-terminal domain and the C-terminal dimerization appendix that are present in MetRSs of several other species. Escherichia coli tRNA^Met was a substrate for the encoded protein as determined by genetic complementation and in vitro aminoacylation reaction. This cross-species aminoacylation activity suggests the conservation of interaction mode between tRNA^Met and MetRS.     

Zinc-induced paracrystalline aggregation of glutamine synthetase

The unique capacity of glutamine synthetase to form highly insoluble paracrystalline aggregates in the presence of Zn²⁺ and Mg²⁺ mixtures is the basis of a new simple procedure for the isolation of the enzyme from crude extracts of Escherichia coli. Under optimal conditions (pH 5.85, 25 °C, 1.5 mm ZnSO₄ and 50 MgCl₂ over 95% of the enzyme is precipitated from crude extracts; differential extraction of the precipitate with dilute buffer (pH 7.0) containing 2.5 mm MgCl₂ leads to high yields of almost pure glutamine synthetase. Polyacrylamide gel electrophoresis of the purified enzyme shows it to consist of one major protein and two minor protein components, all of which exhibit glutamine synthetase activity. The major component appears to be identical with the enzyme previously isolated by the older more tedious procedure of Woolfolk et al. (1966). The γ-glutamyl transferase activity of enzyme isolated by the new procedure is the same as that isolated by the older method, but its biosynthetic activity is 25–35% lower. In all other respects examined (i.e., divalent ion specificity, pH optimum, apparent K_m values for substrates, susceptibility to feedback inhibition and physical properties) enzymes prepared by the old and the new procedures are indistinguishable. From studies with pure glutamine synthetase isolated by either procedure, it has been established that paracrystalline aggregation does not occur until 9–10 equivs of Zn²⁺ are bound per mole of enzyme. The high specificity of Zn²⁺ in inducing enzyme aggregation, suggests that its binding provokes a unique conformational state of the enzyme. This is supported by the fact that addition of Zn²⁺ to relaxed (divalent cation free) enzyme elicits a change in the ultraviolet spectrum of the enzyme that is qualitatively different from that caused by either Mg²⁺ or Mn²⁺. Moreover, in contrast to Mg²⁺, the binding of Zn²⁺ decreases the fluorescence associated with the binding of 2-p-toludinyl-naphthalene-6-sulfonic acid to the enzyme, suggesting that Zn²⁺ binding is accompanied by a decrease in the number of exposed hydrophobic regions on the enzyme.     

Pyrrolysyl-tRNA synthetase variants reveal ancestral aminoacylation function

Pyrrolysyl-tRNA synthetase (PylRS) is a class IIc aminoacyl-tRNA synthetase that is related to phenylalanyl-tRNA synthetase (PheRS). Genetic selection provided PylRS variants with a broad range of specificity for diverse non-canonical amino acids (ncAAs). One variant is a specific phenylalanine-incorporating enzyme. Structural models of the PylRSamino acid complex show that the small pocket size and π-interaction play an important role in specific recognition of Phe and the engineered PylRS active site resembles that of Escherichia coli PheRS.     

A comparative analysis of the fluorescence properties of the wild-type and active site mutants of the hepatitis C virus autoprotease NS2-3

Hepatitis C virus encodes an autoprotease, NS2-3, which is required for processing of the viral polyprotein between the non-structural NS2 and NS3 proteins. This protease activity is vital for the replication and assembly of the virus and therefore represents a target for the development of anti-viral drugs. The mechanism of this auto-processing reaction is not yet clear but the protease activity has been shown to map to the C-terminal region of NS2 and the N-terminal serine protease region of NS3. The NS2-3 precursor can be expressed in Escherichia coli as inclusion bodies, purified as denatured protein and refolded, in the presence of detergents and the divalent metal ion zinc, into an active form capable of auto-cleavage. Here, intrinsic tryptophan fluorescence has been used to assess refolding in the wild-type protein and specific active site mutants. We also investigate the effects on protein folding of alterations to the reaction conditions that have been shown to prevent auto-cleavage. Our data demonstrate that these active site mutations do not solely affect the cleavage activity of the HCV NS2-3 protease but significantly affect the integrity of the global protein fold.     

A conserved chloramphenicol binding site at the entrance to the ribosomal peptide exit tunnel

The antibiotic chloramphenicol produces modifications in 23S rRNA when bound to ribosomes from the bacterium Escherichia coli and the archaeon Halobacterium halobium and irradiated with 365 nm light. The modifications map to nucleotides m⁵U747 and C2611/C2612, in domains II and V, respectively, of E.coli 23S rRNA and G2084 (2058 in E.coli numbering) in domain V of H.halobium 23S rRNA. The modification sites overlap with a portion of the macrolide binding site and cluster at the entrance to the peptide exit tunnel. The data correlate with the recently reported chloramphenicol binding site on an archaeal ribosome and suggest that a similar binding site is present on the E.coli ribosome.