Resilience and proteome response of Escherichia coli to high levels of isoleucine mistranslation.

Accurate pairing of amino acids and tRNAs is a prerequisite for faithful translation of genetic information during protein biosynthesis. Here we present the effects of proteome-wide mistranslation of isoleucine (Ile) by canonical valine (Val) or non-proteinogenic norvaline (Nva) in a genetically engineered Escherichia coli strain with an editing-defective isoleucyl-tRNA synthetase (IleRS). Editing-defective IleRS efficiently mischarges both Val and Nva to tRNAIle and impairs the translational accuracy of Ile decoding. When mistranslation was induced by the addition of Val or Nva to the growth medium, an Ile-to-Val or Ile-to-Nva substitution of up to 20 % was measured by high-resolution mass spectrometry. This mistranslation level impaired bacterial growth, promoted the SOS response and filamentation during stationary phase, caused global proteome dysregulation and upregulation of the cellular apparatus for maintaining proteostasis, including the major chaperones (GroES/EL, DnaK/DnaJ/GrpE and HtpG), the disaggregase ClpB and the proteases (Lon, HslV/HslU, ClpA, ClpS). The most important consequence of mistranslation appears to be non-specific protein aggregation, which is effectively counteracted by the disaggregase ClpB. Our data show that E. coli can sustain high isoleucine mistranslation levels and remain viable despite excessive protein aggregation and severely impaired translational fidelity. However, we show that inaccurate translation lowers bacterial resilience to heat stress and decreases bacterial survival at elevated temperatures.

Structural basis for substrate and antibiotic recognition by Helicobacter pylori isoleucyl-tRNA synthetase.

Helicobacter pylori infection is a global health concern, affecting over half of the world's population. Acquiring structural information on pharmacological targets is crucial to facilitate inhibitor design. Here, we have determined the crystal structures of H. pylori isoleucyl-tRNA synthetase (HpIleRS) in apo form as well as in complex with various substrates (Ile, Ile-AMP, Val, and Val-AMP) or an inhibitor (mupirocin). Our results provide valuable insights into substrate specificity, recognition, and the mechanism by which HpIleRS is inhibited by an antibiotic. Moreover, we identified Asp641 as a prospective regulatory site and conducted biochemical analyses to investigate its regulatory mechanism. The detailed structural information acquired from this research holds promise for the development of highly selective and effective inhibitors against H. pylori infection.

Gly56 in the synthetic site of isoleucyl-tRNA synthetase confers specificity and maintains communication with the editing site.

Isoleucyl-tRNA synthetase (IleRS) links isoleucine to cognate tRNA via the Ile-AMP intermediate. Non-cognate valine is often mistakenly recognized as the IleRS substrate; therefore, to maintain the accuracy of translation, IleRS hydrolyzes Val-AMP within the synthetic site (pre-transfer editing). As this activity is not efficient enough, Val-tRNAIle is formed and hydrolyzed in the distant post-transfer editing site. A strictly conserved synthetic site residue Gly56 was previously shown to safeguard Ile-to-Val discrimination during aminoacyl (aa)-AMP formation. Here, we show that the Gly56Ala variant lost its specificity in pre-transfer editing, confirming that this residue ensures the selectivity of all synthetic site reactions. Moreover, we found that the Gly56Ala mutation affects IleRS interaction with aa-tRNA likely by disturbing tRNA-dependent communication between the two active sites.

Regulation of BRCA1 stability through the tandem UBX domains of isoleucyl-tRNA synthetase 1.

Aminoacyl-tRNA synthetases (ARSs) have evolved to acquire various additional domains. These domains allow ARSs to communicate with other cellular proteins in order to promote non-translational functions. Vertebrate cytoplasmic isoleucyl-tRNA synthetases (IARS1s) have an uncharacterized unique domain, UNE-I. Here, we present the crystal structure of the chicken IARS1 UNE-I complexed with glutamyl-tRNA synthetase 1 (EARS1). UNE-I consists of tandem ubiquitin regulatory X (UBX) domains that interact with a distinct hairpin loop on EARS1 and protect its neighboring proteins in the multi-synthetase complex from degradation. Phosphomimetic mutation of the two serine residues in the hairpin loop releases IARS1 from the complex. IARS1 interacts with BRCA1 in the nucleus, regulates its stability by inhibiting ubiquitylation via the UBX domains, and controls DNA repair function.

Recognition of tRNAIle with a UAU anticodon by isoleucyl-tRNA synthetase in lactic acid bacteria.

In almost all eubacteria, the AUA codon is translated by tRNAIle2 bearing lysidine at the wobble position. Lysidine is introduced by tRNAIle lysidine synthetase (TilS) via post-transcriptional modification of the cytidine of tRNAIle2 (CAU). Lactobacillus casei and Lactobacillus plantarum have tilS homologues and tRNAIle2 (CAU) genes. In addition, L. casei also has another tRNAIle2 gene with an UAU anticodon. L. plantarum has a tRNAIle (UAU)-like RNA. Here, we demonstrate that L. casei tRNAIle2 (UAU) is charged with isoleucine by L. casei isoleucyl-tRNA synthetase (IleRS) but not by L. plantarum IleRS, even though the amino acid identity of these two enzymes is over 60%. It has been reported that, in Mycoplasma mobile, which has its tRNAIle2 (UAU) but no tilS homologue, an Arg residue at position 865 of the IleRS is required for recognition of the UAU anticodon. This position is occupied by an Arg also in the IleRSs from both of the Lactobacillus species. Thus, other residues in L. casei, IleRS should also contribute to the recognition of tRNAIle2 (UAU). We found that a chimeric L. casei IleRS in which the N-terminal domain was replaced by the corresponding region of L. plantatarum IleRS has very low aminoacylation activity towards both tRNAIle2 (UAU) and tRNAIle1 (GAU). The A18G mutant had barely detectable aminoacylation activity towards either of the tRNAsIle . However, a double point mutant of A18G and G19N aminoacylated tRNAIle1 (GAU), but not tRNAIle2 (UAU). Our results suggest that, for L. casei IleRS, Ala18 and Gly19 also play a critical role in recognition of tRNAIle2 (UAU).

Dynamics of the Catalytic Active Site of Isoleucyl tRNA Synthetase from Staphylococcus aureus bound with Adenylate and Mupirocin.

The development of new antimicrobial drugs is critically needed due to the alarming increase in antibiotic resistance in bacterial pathogens. The active sites of different bacterial aminoacyl tRNA synthetases (aaRS) are validated targets of antibiotics. At present, the only aaRS inhibitor approved is mupirocin (MRC) which targets bacterial isoleucyl tRNA synthetase (IleRS). The present work is aimed at understanding the lacunae of knowledge concerning the active site conformational dynamics in IleRS in the presence of inhibitor mupirocin. With this end in view, we have carried out classical molecular dynamics simulation and metadynamics simulations of the open state of IleRS from Staphylococcus aureus (SaIleRS), the closed state tripartite complex bound with cognate adenylate (Ile-AMP) and tRNA, the closed state tripartite complex bound with noncognate MRC, and the closed state tripartite complex bound with tRNA and MRC with mutated SaIleRS (V588F). The present simulation established a dynamic picture of SaIleRS complexed with cognate and the noncognate substrates which are completely consistent with crystallographic and biochemical studies and explain the existing lacunae of knowledge. The active site is significantly more compact in the Ile-AMP bound complex compared to the open state due to the closure of the KMSKS and HMGH loops and clamping down of the tRNA acceptor end near the active site gate. The present result shows that the unusual open conformational state of the KMSKS loop observed in the cognate substrate-bound complex in the crystal is due to crystallographic constraints. Although the mupirocin tightly fits the catalytic active site in the MRC-bound complex, the nonanoic acid moiety is partly exposed to the water. The KMSKS loop is pushed open in the MRC-bound complex to accommodate the noncognate MRC. New tunnels open up, extending to the editing site in the complex. Out of its three broad segments, the C12 to C17 segment, the conjugated segment, and the nonanoic moiety, the conjugated part of MRC binds most effectively with the active site of the MRC-bound complex. The aromatic residues packing around the C12 to C17 segment of MRC stabilize the tRNA hairpin conformation in a similar way as observed in the TrpRS. The V588F mutation is weakening the interaction between this region of the active site and weakens the binding of MRC in the active site. This result explains why the V588F mutation is responsible for low-level mupirocin resistance. The free energy of unbinding the conjugated segment (and C12 to C17 segment, as well) largely contributes to the total free energy of unbinding the MRC. The active site organization of IleRS from eukaryotic Candida albicans is compared with the bacterial IleRS active site to understand the low binding affinity in eukaryotic IleRS. The present study could be a starting point of future studies related to the development of effective drug binding in the SaIleRS.

Inhibitory mechanism of reveromycin A at the tRNA binding site of a class I synthetase.

The polyketide natural product reveromycin A (RM-A) exhibits antifungal, anticancer, anti-bone metastasis, anti-periodontitis and anti-osteoporosis activities by selectively inhibiting eukaryotic cytoplasmic isoleucyl-tRNA synthetase (IleRS). Herein, a co-crystal structure suggests that the RM-A molecule occupies the substrate tRNAIle binding site of Saccharomyces cerevisiae IleRS (ScIleRS), by partially mimicking the binding of tRNAIle. RM-A binding is facilitated by the copurified intermediate product isoleucyl-adenylate (Ile-AMP). The binding assays confirm that RM-A competes with tRNAIle while binding synergistically with L-isoleucine or intermediate analogue Ile-AMS to the aminoacylation pocket of ScIleRS. This study highlights that the vast tRNA binding site of the Rossmann-fold catalytic domain of class I aminoacyl-tRNA synthetases could be targeted by a small molecule. This finding will inform future rational drug design.

Phenyltriazole-functionalized sulfamate inhibitors targeting tyrosyl- or isoleucyl-tRNA synthetase.

Antimicrobial resistance is considered as one of the major threats for the near future as the lack of effective treatments for various infections would cause more deaths than cancer by 2050. The development of new antibacterial drugs is considered as one of the cornerstones to tackle this problem. Aminoacyl-tRNA synthetases (aaRSs) are regarded as good targets to establish new therapies. Apart from being essential for cell viability, they are clinically validated. Indeed, mupirocin, an isoleucyl-tRNA synthetase (IleRS) inhibitor, is already commercially available as a topical treatment for MRSA infections. Unfortunately, resistance developed soon after its introduction on the market, hampering its clinical use. Therefore, there is an urgent need for new cellular targets or improved therapies. Follow-up research by Cubist Pharmaceuticals led to a series of selective and in vivo active aminoacyl-sulfamoyl aryltetrazole inhibitors targeting IleRS (e.g. CB 168). Here, we describe the synthesis of new IleRS and TyrRS inhibitors based on the Cubist Pharmaceuticals compounds, whereby the central ribose was substituted for a tetrahydropyran ring. Various linkers were evaluated connecting the six-membered ring with the base-mimicking part of the synthesized analogues. Out of eight novel molecules, a three-atom spacer to the phenyltriazole moiety, which was established using azide-alkyne click chemistry, appeared to be the optimized linker to inhibit IleRS. However, 11 (Ki,app = 88 ± 5.3 nM) and 36a (Ki,app = 114 ± 13.5 nM) did not reach the same level of inhibitory activity as for the known high-affinity natural adenylate-intermediate analogue isoleucyl-sulfamoyl adenosine (IleSA, CB 138; Ki,app = 1.9 ± 4.0 nM) and CB 168, which exhibit a comparable inhibitory activity as the native ligand. Therefore, 11 was docked into the active site of IleRS using a known crystal structure of T. thermophilus in complex with mupirocin. Here, we observed the loss of the crucial 3'- and 4'- hydroxyl group interactions with the target enzyme compared to CB 168 and mupirocin, which we suggest to be the reason for the limited decrease in enzyme affinity. Despite the lack of antibacterial activity, we believe that structurally optimizing these novel analogues via a structure-based approach could ultimately result in aaRS inhibitors which would help to tackle the antibiotic resistance problem.

Structural Basis for the Antibiotic Resistance of Eukaryotic Isoleucyl-tRNA Synthetase.

Pathogenic aminoacyl-tRNA synthetases (ARSs) are attractive targets for anti-infective agents because their catalytic active sites are different from those of human ARSs. Mupirocin is a topical antibiotic that specifically inhibits bacterial isoleucy-ltRNA synthetase (IleRS), resulting in a block to protein synthesis. Previous studies on Thermus thermophilus IleRS indicated that mupirocin-resistance of eukaryotic IleRS is primarily due to differences in two amino acids, His581 and Leu583, in the active site. However, without a eukaryotic IleRS structure, the structural basis for mupirocin-resistance of eukaryotic IleRS remains elusive. Herein, we determined the crystal structure of Candida albicans IleRS complexed with Ile-AMP at 2.9 Å resolution. The largest difference between eukaryotic and prokaryotic IleRS enzymes is closure of the active site pocket by Phe55 in the HIGH loop; Arg410 in the CP core loop; and the second Lys in the KMSKR loop. The Ile-AMP product is lodged in a closed active site, which may restrict its release and thereby enhance catalytic efficiency. The compact active site also prevents the optimal positioning of the 9-hydroxynonanoic acid of mupirocin and plays a critical role in resistance of eukaryotic IleRS to anti-infective agents.

Mechanism of discrimination of isoleucyl-tRNA synthetase against nonproteinogenic α-aminobutyrate and its fluorinated analogues.

Isoleucyl-tRNA synthetase (IleRS) is a paradigm for understanding how specificity against smaller hydrophobic substrates evolved in both the synthetic and editing reactions. IleRS misactivates nonproteinogenic norvaline (Nva) and proteinogenic valine (Val), with a 200-fold lower efficiency than the cognate isoleucine (Ile). Translational errors are, however, prevented by IleRS hydrolytic editing. Nva and Val are both smaller than Ile by a single methylene group. How does the removal of one additional methylene group affects IleRS specificity? We found that the nonproteinogenic α-aminobutyrate (Abu) is activated 30-fold less efficiently than Nva and Val, indicating that the removal of the second methylene group comes with a lower penalty. As with Nva and Val, discrimination against Abu predominantly originated from a higher KM . To examine whether increased hydrophobicity could compensate for the loss of van der Waals interactions, we tested fluorinated Abu analogues. We found that fluorination further hampered activation by IleRS, and even more so by the evolutionary-related ValRS. This suggests that hydrophobicity is not a main driving force of substrate binding in these enzymes. Finally, a discrimination factor of 7100 suggests that IleRS is not expected to edit Abu. However, we found that the IleRS editing domain hydrolyzes Abu-tRNAIle with a rate of 40 s-1 and the introduction of fluorine did not slow down the hydrolysis. This raises interesting questions regarding the mechanism of specificity of the editing domain and its evolution. Understanding what shapes IleRS specificity is also of importance for reengineering translation to accommodate artificial substrates including fluorinated amino acids. ENZYMES: Isoleucyl-tRNA synthetase (EC 6.1.1.5), leucyl-tRNA synthetase (EC 6.1.1.4), valyl-tRNA synthetase (EC 6.1.1.9).

New tRNA contacts facilitate ligand binding in a Mycobacterium smegmatis T box riboswitch.

T box riboswitches are RNA regulatory elements widely used by organisms in the phyla Firmicutes and Actinobacteria to regulate expression of amino acid-related genes. Expression of T box family genes is down-regulated by transcription attenuation or inhibition of translation initiation in response to increased charging of the cognate tRNA. Three direct contacts with tRNA have been described; however, one of these contacts is absent in a subclass of T box RNAs and the roles of several structural domains conserved in most T box RNAs are unknown. In this study, structural elements of a Mycobacterium smegmatis ileS T box riboswitch variant with an Ultrashort (US) Stem I were sequentially deleted, which resulted in a progressive decrease in binding affinity for the tRNAIle ligand. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) revealed structural changes in conserved riboswitch domains upon interaction with the tRNA ligand. Cross-linking and mutational analyses identified two interaction sites, one between the S-turn element in Stem II and the T arm of tRNAIle and the other between the Stem IIA/B pseudoknot and the D loop of tRNAIle These newly identified RNA contacts add information about tRNA recognition by the T box riboswitch and demonstrate a role for the S-turn and pseudoknot elements, which resemble structural elements that are common in many cellular RNAs.

The tRNA A76 Hydroxyl Groups Control Partitioning of the tRNA-dependent Pre- and Post-transfer Editing Pathways in Class I tRNA Synthetase.

Aminoacyl-tRNA synthetases catalyze ATP-dependent covalent coupling of cognate amino acids and tRNAs for ribosomal protein synthesis. Escherichia coli isoleucyl-tRNA synthetase (IleRS) exploits both the tRNA-dependent pre- and post-transfer editing pathways to minimize errors in translation. However, the molecular mechanisms by which tRNA(Ile) organizes the synthetic site to enhance pre-transfer editing, an idiosyncratic feature of IleRS, remains elusive. Here we show that tRNA(Ile) affects both the synthetic and editing reactions localized within the IleRS synthetic site. In a complex with cognate tRNA, IleRS exhibits a 10-fold faster aminoacyl-AMP hydrolysis and a 10-fold drop in amino acid affinity relative to the free enzyme. Remarkably, the specificity against non-cognate valine was not improved by the presence of tRNA in either of these processes. Instead, amino acid specificity is determined by the protein component per se, whereas the tRNA promotes catalytic performance of the synthetic site, bringing about less error-prone and kinetically optimized isoleucyl-tRNA(Ile) synthesis under cellular conditions. Finally, the extent to which tRNA(Ile) modulates activation and pre-transfer editing is independent of the intactness of its 3'-end. This finding decouples aminoacylation and pre-transfer editing within the IleRS synthetic site and further demonstrates that the A76 hydroxyl groups participate in post-transfer editing only. The data are consistent with a model whereby the 3'-end of the tRNA remains free to sample different positions within the IleRS·tRNA complex, whereas the fine-tuning of the synthetic site is attained via conformational rearrangement of the enzyme through the interactions with the remaining parts of the tRNA body.

Determinants for tRNA-dependent pretransfer editing in the synthetic site of isoleucyl-tRNA synthetase.

The accurate expression of genetic information relies on the fidelity of amino acid-tRNA coupling by aminoacyl-tRNA synthetases (aaRS). When the specificity against structurally similar noncognate amino acids in the synthetic reaction does not support a threshold fidelity level for translation, the aaRS employ intrinsic hydrolytic editing to correct errors in aminoacylation. Escherichia coli isoleucyl-tRNA synthetase (EcIleRS) is a class I aaRS that is notable for its use of tRNA-dependent pretransfer editing to hydrolyze noncognate valyl-adenylate prior to aminoacyl-tRNA formation. On the basis of the finding that IleRS possessing an inactivated post-transfer editing domain is still capable of robust tRNA-dependent editing, we have recently proposed that the pretransfer editing activity resides within the synthetic site. Here we apply an improved methodology that allows quantitation of the AMP fraction that arises particularly from tRNA-dependent aa-AMP hydrolysis. By this approach, we demonstrate that tRNA-dependent pretransfer editing accounts for nearly one-third of the total proofreading by EcIleRS and that a highly conserved tyrosine within the synthetic site modulates both editing and aminoacylation. Therefore, synthesis of aminoacyl-tRNA and hydrolysis of aminoacyl-adenylates employ overlapping amino acid determinants. We suggest that this overlap hindered the evolution of synthetic site-based pretransfer editing as the predominant proofreading pathway, because that activity is difficult to accommodate in the context of efficient aminoacyl-tRNA synthesis. Instead, the acquisition of a spatially separate domain dedicated to post-transfer editing alone allowed for the development of a powerful deacylation machinery that effectively competes with dissociation of misacylated tRNAs.

Mutation in the nuclear-encoded mitochondrial isoleucyl-tRNA synthetase IARS2 in patients with cataracts, growth hormone deficiency with short stature, partial sensorineural deafness, and peripheral neuropathy or with Leigh syndrome.

Mutations in the nuclear-encoded mitochondrial aminoacyl-tRNA synthetases are associated with a range of clinical phenotypes. Here, we report a novel disorder in three adult patients with a phenotype including cataracts, short-stature secondary to growth hormone deficiency, sensorineural hearing deficit, peripheral sensory neuropathy, and skeletal dysplasia. Using SNP genotyping and whole-exome sequencing, we identified a single likely causal variant, a missense mutation in a conserved residue of the nuclear gene IARS2, encoding mitochondrial isoleucyl-tRNA synthetase. The mutation is homozygous in the affected patients, heterozygous in carriers, and absent in control chromosomes. IARS2 protein level was reduced in skin cells cultured from one of the patients, consistent with a pathogenic effect of the mutation. Compound heterozygous mutations in IARS2 were independently identified in a previously unreported patient with a more severe mitochondrial phenotype diagnosed as Leigh syndrome. This is the first report of clinical findings associated with IARS2 mutations.

Kinetic proofreading at single molecular level: aminoacylation of tRNA(Ile) and the role of water as an editor.

Proofreading/editing in protein synthesis is essential for accurate translation of information from the genetic code. In this article we present a theoretical investigation of efficiency of a kinetic proofreading mechanism that employs hydrolysis of the wrong substrate as the discriminatory step in enzyme catalytic reactions. We consider aminoacylation of tRNA(Ile) which is a crucial step in protein synthesis and for which experimental results are now available. We present an augmented kinetic scheme and then employ methods of stochastic simulation algorithm to obtain time dependent concentrations of different substances involved in the reaction and their rates of formation. We obtain the rates of product formation and ATP hydrolysis for both correct and wrong substrates (isoleucine and valine in our case, respectively), in single molecular enzyme as well as ensemble enzyme kinetics. The present theoretical scheme correctly reproduces (i) the amplitude of the discrimination factor in the overall rates between isoleucine and valine which is obtained as (1.8×10(2)).(4.33×10(2)) = 7.8×10(4), (ii) the rates of ATP hydrolysis for both Ile and Val at different substrate concentrations in the aminoacylation of tRNA(Ile). The present study shows a non-michaelis type dependence of rate of reaction on tRNA(Ile) concentration in case of valine. The overall editing in steady state is found to be independent of amino acid concentration. Interestingly, the computed ATP hydrolysis rate for valine at high substrate concentration is same as the rate of formation of Ile-tRNA(Ile) whereas at intermediate substrate concentration the ATP hydrolysis rate is relatively low. We find that the presence of additional editing domain in class I editing enzyme makes the kinetic proofreading more efficient through enhanced hydrolysis of wrong product at the editing CP1 domain.

Mapping and exome sequencing identifies a mutation in the IARS gene as the cause of hereditary perinatal weak calf syndrome.

We identified an IARS (isoleucyl-tRNA synthetase) c.235G>C (p.Val79Leu) substitution as the causative mutation for neonatal weakness with intrauterine growth retardation (perinatal weak calf syndrome). In Japanese Black cattle, the syndrome was frequently found in calves sired by Bull A. Hence, we employed homozygosity mapping and linkage analysis. In order to identify the perinatal weak calf syndrome locus in a 4.04-Mb region of BTA 8, we analysed a paternal half-sibling family with a BovineSNP50 BeadChip and microsatellites. In this critical region, we performed exome sequencing to identify a causative mutation. Three variants were detected as possible candidates for causative mutations that were predicted to disrupt the protein function, including a G>C (p.Val79Leu) mutation in IARS c.235. The IARS c.235G>C mutation was not a homozygous risk allele in the 36 healthy offspring of Bull A. Moreover, the IARS Val79 residue and its flanking regions were evolutionarily and highly conserved. The IARS mutant (Leu79) had decreased aminoacylation activity. Additionally, the homozygous mutation was not found in any of 1526 healthy cattle. Therefore, we concluded that the IARS c.235G>C mutation was the cause of hereditary perinatal weak calf syndrome.

Decoding system for the AUA codon by tRNAIle with the UAU anticodon in Mycoplasma mobile.

Deciphering the genetic code is a fundamental process in all living organisms. In many bacteria, AUA codons are deciphered by tRNA(Ile2) bearing lysidine (L) at the wobble position. L is a modified cytidine introduced post-transcriptionally by tRNA(Ile)-lysidine synthetase (TilS). Some bacteria, including Mycoplasma mobile, do not carry the tilS gene, indicating that they have established a different system to decode AUA codons. In this study, tRNA(Ile2) has been isolated from M. mobile and was found to contain a UAU anticodon without any modification. Mycoplasma mobile isoleucyl-tRNA synthetase (IleRS) recognized the UAU anticodon, whereas Escherichia coli IleRS did not efficiently aminoacylate tRNA(Ile2)(UAU). In M. mobile IleRS, a single Arg residue at position 865 was critical for specificity for the UAU anticodon and, when the corresponding site (W905) in E. coli IleRS was substituted with Arg, the W905R mutant efficiently aminoacylated tRNA with UAU anticodon. Mycoplasma mobile tRNA(Ile2) cannot distinguish between AUA and AUG codon on E. coli ribosome. However, on M. mobile ribosome, M. mobile tRNA(Ile2)(UAU) specifically recognized AUA codon, and not AUG codon, suggesting M. mobile ribosome has a property that prevents misreading of AUG codon. These findings provide an insight into the evolutionary reorganization of the AUA decoding system.

Space-related pharma-motifs for fast search of protein binding motifs and polypharmacological targets.

BACKGROUND: To discover a compound inhibiting multiple proteins (i.e. polypharmacological targets) is a new paradigm for the complex diseases (e.g. cancers and diabetes). In general, the polypharmacological proteins often share similar local binding environments and motifs. As the exponential growth of the number of protein structures, to find the similar structural binding motifs (pharma-motifs) is an emergency task for drug discovery (e.g. side effects and new uses for old drugs) and protein functions. RESULTS: We have developed a Space-Related Pharmamotifs (called SRPmotif) method to recognize the binding motifs by searching against protein structure database. SRPmotif is able to recognize conserved binding environments containing spatially discontinuous pharma-motifs which are often short conserved peptides with specific physico-chemical properties for protein functions. Among 356 pharma-motifs, 56.5% interacting residues are highly conserved. Experimental results indicate that 81.1% and 92.7% polypharmacological targets of each protein-ligand complex are annotated with same biological process (BP) and molecular function (MF) terms, respectively, based on Gene Ontology (GO). Our experimental results show that the identified pharma-motifs often consist of key residues in functional (active) sites and play the key roles for protein functions. The SRPmotif is available at http://gemdock.life.nctu.edu.tw/SRP/. CONCLUSIONS: SRPmotif is able to identify similar pharma-interfaces and pharma-motifs sharing similar binding environments for polypharmacological targets by rapidly searching against the protein structure database. Pharma-motifs describe the conservations of binding environments for drug discovery and protein functions. Additionally, these pharma-motifs provide the clues for discovering new sequence-based motifs to predict protein functions from protein sequence databases. We believe that SRPmotif is useful for elucidating protein functions and drug discovery.

Crystallization and preliminary X-ray diffraction analysis of an archaeal tRNA-modification enzyme, TiaS, complexed with tRNA(Ile2) and ATP.

The cytidine at the first anticodon position of archaeal tRNA(Ile2), which decodes the isoleucine AUA codon, is modified to 2-agmatinylcytidine (agm(2)C) to guarantee the fidelity of protein biosynthesis. This post-transcriptional modification is catalyzed by tRNA(Ile)-agm(2)C synthetase (TiaS) using ATP and agmatine as substrates. Archaeoglobus fulgidus TiaS was overexpressed in Escherichia coli cells and purified. tRNA(Ile2) was prepared by in vitro transcription with T7 RNA polymerase. TiaS was cocrystallized with both tRNA(Ile2) and ATP by the vapour-diffusion method. The crystals of the TiaS-tRNA(Ile2)-ATP complex diffracted to 2.9 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the primitive hexagonal space group P3(2)21, with unit-cell parameters a = b = 131.1, c = 86.6 Å. The asymmetric unit is expected to contain one TiaS-tRNA(Ile2)-ATP complex, with a Matthews coefficient of 2.8 Å(3) Da(-1) and a solvent content of 61%.

Biogenesis of 2-agmatinylcytidine catalyzed by the dual protein and RNA kinase TiaS.

The archaeal AUA-codon specific tRNA(Ile) contains 2-agmatinylcytidine (agm(2)C or agmatidine) at the anticodon wobble position (position 34). The formation of this essential modification is catalyzed by tRNA(Ile)-agm(2)C synthetase (TiaS) using agmatine and ATP as substrates. TiaS has a previously unknown catalytic domain, which we have named the Thr18-Cyt34 kinase domain (TCKD). Biochemical analyses of Archaeoglobus fulgidus TiaS and its mutants revealed that the TCKD first hydrolyzes ATP into AMP and pyrophosphate, then phosphorylates the C2 position of C34 with the γ-phosphate. Next, the amino group of agmatine attacks this position to release the phosphate and form agm(2)C. Notably, the TCKD also autophosphorylates the Thr18 of TiaS, which may be involved in agm(2)C formation. Thus, the unique kinase domain of TiaS catalyzes dual phosphorylation of protein and RNA substrates.