Mechanism and evolution of multidomain aminoacyl-tRNA synthetases revealed by their inhibition by analogues of a reaction intermediate, and by properties of truncated forms

Many enzymes which catalyze the conversion of large substrates are made of several structural domains belonging to the same polypeptide chain. Transfer RNA (tRNA), one of the substrates of the multidomain aminoacyl-tRNA synthetases (aaRS), is an L-shaped molecule whose size in one dimension is similar to that of its cognate aaRS. Crystallographic structures of aaRS/tRNA complexes show that these enzymes use several of their structural domains to interact with their cognate tRNA. This mini review discusses first some aspects of the evolution and of the flexibility of the pentadomain bacterial glutamyl-tRNA synthetase (GluRS) revealed by kinetic and interaction studies of complementary truncated forms, and then illustrates how stable analogues of aminoacyl-AMP intermediates have been used to probe conformational changes in the active sites of Escherichia coli GluRS and of the nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) of Pseudomonas aeruginosa.

The Role of Anionic Protein Residues on the Salt Dependence of the Binding of Aminoacyl-tRNA Synthetases to tRNA:A Poisson-Boltzmann Analysis

Long-range electrostatic interactions in proteins/peptides associating to nucleic acids are reflected in the salt-dependence of the binding process.According to the oligocationic binding model,which is based on counterion condensation theory,only the cationic residues of peptides/proteins near the binding interface are assumed to affect the salt dependence in the association of peptides and proteins to nucleic acids.This model has been used to interpret and predict the binding of oligocationic chains-such as oligoarginines/lysines-to nucleic acids,and does an excellent job in these kinds of systems.This simple relationship,which is used to compare or count the number of ionic interactions in protein-nucleic acid complexes,does not hold when acidic residues,i.e.glutamate and aspartate,are incorporated in the protein matrix.Here,we report a combined molecular mechanics(by means of energy-minimization of the structure under the influence of an empirical energy function)and Poisson-Boltzmann(PB)study on the salt-dependence in binding to tRNA of two important enzymes that are involved in the seminal step of peptide formation in the ribosome:Glutamine synthetase(GluRS)and Glutaminyl synthetase(GlnRS)bound to their cognate tRNA.These two proteins are anionic and contain a significant number of acidic residues distributed over the entire protein.Some of these residues are located in the binding interface to tRNA.We computed the salt-dependence in association,SKpred,of these enzyme-tRNA complexes using both the linear and nonlinear solution to the PoissonBoltzmann Equation(PBE).Our findings demonstrate that the SKpred obtained with the nonlinear PBE is in good agreement with the experimental SKobs,while use of the linear PBE resulted in the SKpred being anomalous.We conclude that electrostatic interactions between the binding partners in these systems are less favorable by means of charge-charge repulsion between negatively charged protein residues and phosphateoxygens in the tRNA backbone but also play a significant role in the association process of proteins to tRNA.Some unfavorable electrostatic interactions are probably compensated by hydrogen-bonds between the carboxylate group of the side chain in the interfacial acidic protein residues and the tRNA backbone.We propose that the low experimentally observed SKobs values for both GlnRS-and GluRS-tRNA depend on the distribution and number of anionic residues that exist in these tRNA synthetases.Our computed electrostatic binding free energies were large and unfavorable due to the Coulombic and de-solvation contribution for the GlnRS-tRNA and GluRS-tRNA complexes,respectively.Thus,low SKobs values may not reflect small contributions from the electrostatic contribution in complex-formation,as is often suggested in the literature.When charges are”turned off”in a computer-experiment,our results indicated that”turning off”acidic residues far from a phosphate group significantly influences SKpred.If cationic residues are“turned off”,less impact on SKpred is observed with respect to the distance to the nearest phosphate-group。

Dual Targeting to Mitochondria and Chloroplasts： Characterization of Thr-tRNA Synthetase Targeting Peptide

There is a group of proteins that are encoded by a single gene, expressed as a single precursor protein and dually targeted to both mitochondria and chloroplasts using an ambiguous targeting peptide. Sequence analysis of 43 dual targeted proteins in comparison with 385 mitochondrial proteins and 567 chloroplast proteins ofArabidopsis thaliana revealed an overall significant increase in phenylalanines, leucines, and serines and a decrease in acidic amino acids and glycine in dual targeting peptides （dTPs）. The N-terminal portion of dTPs has significantly more serines than mTPs. The number of arginines is similar to those in mTPs, but almost twice as high as those in cTPs. We have investigated targeting determinants of the dual targeting peptide of Thr-tRNA synthetase （ThrRS-dTP） studying organellar import of N- and C-terminal deletion constructs of ThrRS-dTP coupled to GFR These results show that the 23 amino acid long N-terminal portion of ThrRS-dTP is crucial but not sufficient for the organellar import. The C-terminal deletions revealed that the shortest peptide that was capable of conferring dual targeting was 60 amino acids long. We have purified the ThrRS- dTP（2-60） to homogeneity after its expression as a fusion construct with GST followed by CNBr cleavage and ion exchange chromatography. The purified ThrRS-dTP（2-60） inhibited import of pF1β into mitochondria and of pSSU into chloroplasts at μM concentrations showing that dual and organelle-specific proteins use the same organellar import pathways. Furthermore, the CD spectra of ThrRS-dTP（2-60） indicated that the peptide has the propensity for forming α-helical structure in membrane mimetic environments; however, the membrane charge was not important for the amount of induced helical structure. This is the first study in which a dual targeting peptide has been purified and investigated by biochemical and biophysical means.

Amino Acid Sensing Using an Ion-Sensitive Field-Effect Transistor

Ion-sensitive field-effect transistor （ISFET）-based biosensor for amino acids was proposed, and the response of the sensor to amino acids was evaluated. As a molecular recognition element, aminoacyl-tRNA synthetase which would be expected to have high selectivity for the corresponding amino acids was used, aminoacyl-tRNA synthetase was coated onto an ISFET electrode, and the response of the biosensor to amino acids was evaluated. The amino acid sensor for tyrosine showed a selective response to tyrosine because of the specific binding ability of aminoacyl-tRNA synthetase for tyrosine and from 300μM to 900 μM of tyrosine could be measured （r2 〉 0.969）.

VARS2 gene mutation leading to overall developmental delay in a child with epilepsy: A case report

BACKGROUND The mitochondrial respiratory chain defects have become the most common cause of neurometabolic disorders in children and adults,which can occur at any time in life,often associated with neurological dysfunction,and lead to chronic disability and premature death.Approximately one-third of patients with mitochondrial disease have biochemical defects involving multiple respiratory chain complexes,suggesting defects in protein synthesis within the mitochondria.We here report a child with VARS2 gene mutations causing mitochondrial disease.CASE SUMMARY A girl,aged 3 years and 4 mo,had been unable to sit and crawl alone since birth,with obvious seizures and microcephaly.Brain magnetic resonance imaging showed symmetrical,flaky,long T1-weighted and low T2-weighted signals in the posterior part of the bilateral putamen with a high signal shadow.T2 fluidattenuated inversion recovery imaging showed a slightly high signal and diffusion-weighted imaging showed an obvious high signal.Whole-exome gene sequencing revealed a compound heterozygous mutation in the VARS2 gene,c.1163(exon11)C>T and c.1940(exon20)C>T,which was derived from the parents.The child was diagnosed with combined oxidative phosphorylation deficiency type 20.CONCLUSION In this patient,mitochondrial disorders including Leigh syndrome and MELAS syndrome(mitochondrial myopathy,encephalopathy,lactic acidosis,and stroke-like episodes)were ruled out,and combined oxidative phosphorylation deficiency type 20 was diagnosed,expanding the phenotypic spectrum of the disease.

谷氨酰-脯氨酰-tRNA合成酶(EPRS)结构与功能的新认识

氨基酰-tRNA合成酶(ARS)催化特异的氨基酸结合到对应tRNA上,确保mRNA高精度地翻译为蛋白质。最近陆续报道ARS同时也在细胞基本生命活动如RNA运输、转录、翻译、免疫反应以及血管生成中充当重要的调节分子和活性成分。目前报道谷氨酰-脯氨酰-tRNA合成酶(EPRS)在具有催化谷氨酸和脯氨酸分别结合到不同tRNA上的功能之外,还可通过基因选择性沉默机制阻遏血浆铜蓝蛋白的表达,从而终止炎症反应等。本文就该酶结构与功能的研究新进展进行综述,并对其进行分子进化分析。

Transfer RNA:A dancer between charging and mis-charging for protein biosynthesis

Transfer RNA plays a fundamental role in the protein biosynthesis as an adaptor molecule by functioning as a biological link between the genetic nucleotide sequence in the mRNA and the amino acid sequence in the protein.To perform its role in protein biosynthesis,it has to be accurately recognized by aminoacyl-tRNA synthetases(aaRSs)to generate aminoacyl-tRNAs(aa-tRNAs).The correct pairing between an amino acid with its cognate tRNA is crucial for translational quality control.Production and utilization of mis-charged tRNAs are usually detrimental for all the species,resulting in cellular dysfunctions.Correct aa-tRNAs formation is collectively controlled by aaRSs with distinct mechanisms and/or other trans-factors.However,in very limited instances,mis-charged tRNAs are intermediate for specific pathways or essential components for the translational machinery.Here,from the point of accuracy in tRNA charging,we review our understanding about the mechanism ensuring correct aa-tRNA generation.In addition,some unique mis-charged tRNA species necessary for the organism are also briefly described.

Hearing impairment-associated KARS mutations lead to defects in aminoacylation of both cytoplasmic and mitochondrial tRNA^(Lys)

Aminoacyl-tRNA synthetases(aaRSs)are ubiquitously expressed,essential enzymes,synthesizing aminoacyl-tRNAs for protein synthesis.Functional defects of aaRSs frequently cause various human disorders.Human KARS encodes both cytosolic and mitochondrial lysyl-tRNA synthetases(LysRSs).Previously,two mutations(c.1129 G>A and c.517 T>C)were identified that led to hearing impairment;however,the underlying biochemical mechanism is unclear.In the present study,we found that the two mutations have no impact on the incorporation of LysRS into the multiple-synthetase complex in the cytosol,but affect the cytosolic LysRS level,its tertiary structure,and cytosolic tRNA aminoacylation in vitro.As for mitochondrial translation,the two mutations have little effect on the steady-state level,mitochondrial targeting,and tRNA binding affinity of mitochondrial LysRS.However,they exhibit striking differences in charging mitochondrial tRNALys,with the c.517T>C mutant being completely deficient in vitro and in vivo.We constructed two yeast genetic models,which are powerful tools to test the in vivo aminoacylation activity of KARS mutations at both the cytosolic and mitochondrial levels.Overall,our data provided biochemical insights into the potentially molecular pathological mechanism of KARS c.1129G>A and c.517T>C mutations and provided yeast genetic bases to investigate other KARS mutations in the future.

Distinct pathogenic mechanisms of various RARS1 mutations in Pelizaeus-Merzbacher-like disease

Mutations of the genes encoding aminoacyl-tRNA synthetases are highly associated with various central nervous system disorders.Recurrent mutations,including c.5A>G,p.D2G;c.1367C>T,p.S456L;c.1535G>A,p.R512Q and c.1846_1847del,p.Y616Lfs*6 of RARS1 gene,which encodes two forms of human cytoplasmic arginyl-tRNA synthetase(hArgRS),are linked to Pelizaeus-Merzbacher-like disease(PMLD)with unclear pathogenesis.Among these mutations,c.5A>G is the most extensively reported mutation,leading to a p.D2G mutation in the N-terminal extension of the long-form hArgRS.Here,we showed the detrimental effects of R512Q substitution andΔC mutations on the structure and function of hArgRS,while the most frequent mutation c.5A>G,p.D2G acted in a different manner without impairing hArgRS activity.The nucleotide substitution c.5A>G reduced translation of hArgRS mRNA,and an upstream open reading frame contributed to the suppressed translation of the downstream main ORF.Taken together,our results elucidated distinct pathogenic mechanisms of various RARS1 mutations in PMLD.