The long-range electrostatic interactions control tRNA-aminoacyl-tRNA synthetase complex formation

In most cases aminoacyl-tRNA synthetases (aaRSs) are negatively charged, as are the tRNA substrates. It is apparent that there are driving forces that provide a long-range attraction between like charge aaRS and tRNA, and ensure formation of "close encounters." Based on numerical solutions to the nonlinear Poisson-Boltzmann equation, we evaluated the electrostatic potential generated by different aaRSs. The 3D-isopotential surfaces calculated for different aaRSs at 0.01 kT/e contour level reveal the presence of large positive patches-one patch for each tRNA molecule. This is true for classes I and II monomers, dimers, and heterotetramers. The potential maps keep their characteristic features over a wide range of contour levels. The results suggest that nonspecific electrostatic interactions are the driving forces of primary stickiness of aaRSs-tRNA complexes. The long-range attraction in aaRS-tRNA systems is explained by capture of negatively charged tRNA into "blue space area" of the positive potential generated by aaRSs. Localization of tRNA in this area is a prerequisite for overcoming the barrier of Brownian motion.     

Single amino acid changes in AspRS reveal alternative routes for expanding its tRNA repertoire in vivo

Aminoacyl-tRNA synthetases (aaRSs) are enzymes that are highly specific for their tRNA substrates. Here, we describe the expansion of a class IIb aaRS-tRNA specificity by a genetic selection that involves the use of a modified tRNA displaying an amber anticodon and the argE(amber) and lacZ(amber) reporters. The study was performed on Escherichia coli aspartyl-tRNA synthetase (AspRS) and amber tRNA(Asp). Nine AspRS mutants able to charge the amber tRNA(Asp) and to suppress the reporter genes were selected from a randomly mutated library. All the mutants exhibited a new amber tRNA(Asp) specificity in addition to the initial native tRNA(Asp). Six mutations were found in the anticodon-binding site located in the N-terminal OB-fold. The strongest suppressor was a mutation of residue Glu-93 that contacts specifically the anticodon nucleotide 34 in the crystal structure. The other mutations in the OB-fold were found at close distance from the anticodon in the so-called loop L45 and strand S1. They concern residues that do not contact tRNA(Asp) in the native complex. In addition, this study shows that suppressors can carry mutations located far from the anticodon-binding site. One such mutation was found in the synthetase hinge-module where it increases the tRNA(Asp)-charging rate, and two other mutations were found in the prokaryotic-specific insertion domain and the catalytic core. These mutants seem to act by indirect effects on the tRNA acceptor stem binding and on the conformation of the active site of the enzyme. Altogether, these data suggest the existence of various ways for modifying the mechanism of tRNA discrimination.     

Evolution meets disease: penetrance and functional epistasis of mitochondrial tRNA mutations

About half of the mitochondrial DNA (mtDNA) mutations causing diseases in humans occur in tRNA genes. Particularly intriguing are those pathogenic tRNA mutations than can reach homoplasmy and yet show very different penetrance among patients. These mutations are scarce and, in addition to their obvious interest for understanding human pathology, they can be excellent experimental examples to model evolution and fixation of mitochondrial tRNA mutations. To date, the only source of this type of mutations is human patients. We report here the generation and characterization of the first mitochondrial tRNA pathological mutation in mouse cells, an m.3739G>A transition in the mitochondrial mt-Ti gene. This mutation recapitulates the molecular hallmarks of a disease-causing mutation described in humans, an m.4290T>C transition affecting also the human mt-Ti gene. We could determine that the pathogenic molecular mechanism, induced by both the mouse and the human mutations, is a high frequency of abnormal folding of the tRNA(Ile) that cannot be charged with isoleucine. We demonstrate that the cells harboring the mouse or human mutant tRNA have exacerbated mitochondrial biogenesis triggered by an increase in mitochondrial ROS production as a compensatory response. We propose that both the nature of the pathogenic mechanism combined with the existence of a compensatory mechanism can explain the penetrance pattern of this mutation. This particular behavior can allow a scenario for the evolution of mitochondrial tRNAs in which the fixation of two alleles that are individually deleterious can proceed in two steps and not require the simultaneous mutation of both.     

与耳聋相关的线粒体tRNA突变

线粒体tRNA基因突变是导致感音神经性耳聋的原因之一．有些tRNA突变可直接造成耳聋的发生,称之为原发突变．如tRNA^Leu(UUR) A3243G等突变与综合征型耳聋相关,而tRNA^Ser(UCN) T7511C等突变则与非综合征型耳聋相关．此外,继发突变如tRNA^Thr G15927A等突变则对原发突变起协同作用,影响耳聋的表型表达．这些突变可引起tRNA二级结构改变,从而影响线粒体蛋白质合成,降低细胞内ATP的产生,由此引起的线粒体功能障碍可导致耳聋的发生．主要讨论与耳聋相关的线粒体tRNA突变及其致聋机理．     

Electrostatic potential of aminoacyl-tRNA synthetase navigates tRNA on its pathway to the binding site

In the first stage of a diffusion-controlled enzymatic reaction, aminoacyl-tRNA synthetases (aaRSs) interact with cognate tRNAs forming non-specific encounters. The aaRSs catalyzing the same overall aminoacylation reaction vary greatly in subunit organization, structural domain composition and amino acid sequence. The diffusional association of aaRS and tRNA was found to be governed by long-range electrostatic interactions when the homogeneous negative potential of tRNA fits to the patches of positive potential produced by aaRS; one patch for each tRNA substrate molecule. Considering aaRS as a molecule with anisotropic reactivity and on the basis of continuum electrostatics and Smoluchowski's theory, the reaction conditions for tRNA-aaRS diffusional encounters were formulated. The domains, categorized as enzymatically relevant, appeared to be non-essential for field sculpturing at long distances. On the other hand, a set of complementary domains exerts primary control on the aaRS isopotential surface formation. Subdividing the aaRS charged residues into native, conservative and non-conservative subsets, we evaluated the contribution of each group to long-range electrostatic potential. Surprisingly, the electrostatic potential landscapes generated by native and non-conservative subsets are fairly similar, thus suggesting the non-conservative subset is developed specifically for efficient tRNA attraction.     

Hearing impairment-associated KARS mutations lead to defects in aminoacylation of both cytoplasmic and mitochondrial tRNALys

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 GA and c.517 TC) 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 tRNA~(Lys), with the c.517TC 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.1129GA and c.517TC mutations and provided yeast genetic bases to investigate other KARS mutations in the future.     

Evolutionary and structural annotation of disease-associated mutations in human aminoacyl-tRNA synthetases

Background

Mutation(s) in proteins are a natural byproduct of evolution but can also cause serious diseases. Aminoacyl-tRNA synthetases (aaRSs) are indispensable components of all cellular protein translational machineries, and in humans they drive translation in both cytoplasm and mitochondria. Mutations in aaRSs have been implicated in a plethora of diseases including neurological conditions, metabolic disorders and cancer.

Results

We have developed an algorithmic approach for genome-wide analyses of sequence substitutions that combines evolutionary, structural and functional information. This pipeline enabled us to super-annotate human aaRS mutations and analyze their linkage to health disorders. Our data suggest that in some but not all cases, aaRS mutations occur in functional and structural sectors where they can manifest their pathological effects by altering enzyme activity or causing structural instability. Further, mutations appear in both solvent exposed and buried regions of aaRSs indicating that these alterations could lead to dysfunctional enzymes resulting in abnormal protein translation routines by affecting inter-molecular interactions or by disruption of non-bonded interactions. Overall, the prevalence of mutations is much higher in mitochondrial aaRSs, and the two most often mutated aaRSs are mitochondrial glutamyl-tRNA synthetase and dual localized glycyl-tRNA synthetase. Out of 63 mutations annotated in this work, only 12 (~20%) were observed in regions that could directly affect aminoacylation activity via either binding to ATP/amino-acid, tRNA or by involvement in dimerization. Mutations in structural cores or at potential biomolecular interfaces account for ~55% mutations while remaining mutations (~25%) remain structurally un-annotated.

Conclusion

This work provides a comprehensive structural framework within which most defective human aaRSs have been structurally analyzed. The methodology described here could be employed to annotate mutations in other protein families in a high-throughput manner.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1063) contains supplementary material, which is available to authorized users.     

Peripheral neuropathy via mutant tRNA synthetases: Inhibition of protein translation provides a possible explanation

Recent evidence indicates that inhibition of protein translation may be a common pathogenic mechanism for peripheral neuropathy associated with mutant tRNA synthetases (aaRSs). aaRSs are enzymes that ligate amino acids to their cognate tRNA, thus catalyzing the first step of translation. Dominant mutations in five distinct aaRSs cause Charcot‐Marie‐Tooth (CMT) peripheral neuropathy, characterized by length‐dependent degeneration of peripheral motor and sensory axons. Surprisingly, loss of aminoacylation activity is not required for mutant aaRSs to cause CMT. Rather, at least for some mutations, a toxic‐gain‐of‐function mechanism underlies CMT‐aaRS. Interestingly, several mutations in two distinct aaRSs were recently shown to inhibit global protein translation in Drosophila models of CMT‐aaRS, by a mechanism independent of aminoacylation, suggesting inhibition of translation as a common pathogenic mechanism. Future research aimed at elucidating the molecular mechanisms underlying the translation defect induced by CMT‐mutant aaRSs should provide novel insight into the molecular pathogenesis of these incurable diseases.     

Transfer RNAs: electrostatic patterns and an early stage of recognition by synthetases and elongation factor EF-Tu

Distributions of phosphate backbone-produced electrostatic potentials around several tRNAs were calculated by solving the nonlinear Poisson-Boltzmann equation. The tRNAs were either free or bound to the proteins involved in translation: aminoacyl-tRNA and elongation factor EF-Tu. We identified several regions of strong negative potential related to typical structural patterns of tRNA and invariant throughout the tRNAs. The patterns are conserved upon binding of tRNAs to the synthetase and the EF-Tu. Variation of tRNA charge in our theoretical calculations of electrostatic potential-mediated pK shifts of pH-dependent labels attached to tRNA, compared to experimentally observed pK shifts for those labels, shows that the total charge of tRNA is large, within the interval of -40 to -70 proton charges. The electrostatic field of tRNA is sufficient to cause ionization of histidine residues of ARSase, causing additional free energy of ARSase-tRNA interaction of at least several kcal/mol. This may discriminate proteins with respect to the particular tRNA at large distances. Two types of tRNA-protein electrostatic recognition mechanisms are discussed. One, more specific, involves charges induced on protein by the large electrostatic potential of tRNA, while the other, less specific, does not involve induced charges.     

人类线粒体tRNA生物合成与线粒体疾病

线粒体是普遍存在于真核细胞中的一类细胞器.每个线粒体含有多个拷贝的闭合环状双链DNA. 人类线粒体DNA (mitochondrial DNA, mtDNA)共编码22种线粒体tRNA(mitochondrial tRNA,mt tRNA), 2种rRNA 及13种多肽.mt tRNA独特的结构特点决定了它们与具有典型三叶草结构的细胞质 tRNA不同.编码mt tRNA的基因突变频率较高,这可能是引起线粒体功能障碍的主要原因之一. 同时 ,这与很多病理现象相关.目前发现,大量与mt tRNA生物代谢和功能相关的核因子包括加工内切酶、 tRNA修饰酶和氨酰-tRNA合成酶.这些核因子的异常导致了疾病相关的tRNA致病突变.由此可见mt tRNA功能对于线粒体活性的重要性.     

Role of tRNA amino acid-accepting end in aminoacylation and its quality control

Aminoacyl–tRNA synthetases (aaRSs) are remarkable enzymes that are in charge of the accurate recognition and ligation of amino acids and tRNA molecules. The greatest difficulty in accurate aminoacylation appears to be in discriminating between highly similar amino acids. To reduce mischarging of tRNAs by non-cognate amino acids, aaRSs have evolved an editing activity in a second active site to cleave the incorrect aminoacyl–tRNAs. Editing occurs after translocation of the aminoacyl–CCA₇₆ end to the editing site, switching between a hairpin and a helical conformation for aminoacylation and editing. Here, we studied the consequence of nucleotide changes in the CCA₇₆ accepting end of tRNA^Leu during the aminoacylation and editing reactions. The analysis showed that the terminal A₇₆ is essential for both reactions, suggesting that critical interactions occur in the two catalytic sites. Substitutions of C₇₄ and C₇₅ selectively decreased aminoacylation keeping nearly unaffected editing. These mutations might favor the regular helical conformation required to reach the editing site. Mutating the editing domain residues that contribute to CCA₇₆ binding reduced the aminoacylation fidelity leading to cell-toxicity in the presence of non-cognate amino acids. Collectively, the data show how protein synthesis quality is controlled by the CCA₇₆ homogeneity of tRNAs.     

Structure of human tryptophanyl-tRNA synthetase in complex with tRNATrp reveals the molecular basis of tRNA recognition and specificity

Aminoacyl-tRNA synthetases (aaRSs) are a family of enzymes responsible for the covalent link of amino acids to their cognate tRNAs. The selectivity and species-specificity in the recognitions of both amino acid and tRNA by aaRSs play a vital role in maintaining the fidelity of protein synthesis. We report here the first crystal structure of human tryptophanyl-tRNA synthetase (hTrpRS) in complex with tRNA^Trp and Trp which, together with biochemical data, reveals the molecular basis of a novel tRNA binding and recognition mechanism. hTrpRS recognizes the tRNA acceptor arm from the major groove; however, the 3′ end CCA of the tRNA makes a sharp turn to bind at the active site with a deformed conformation. The discriminator base A73 is specifically recognized by an α-helix of the unique N-terminal domain and the anticodon loop by an α-helix insertion of the C-terminal domain. The N-terminal domain appears to be involved in Trp activation, but not essential for tRNA binding and acylation. Structural and sequence comparisons suggest that this novel tRNA binding and recognition mechanism is very likely shared by other archaeal and eukaryotic TrpRSs, but not by bacterial TrpRSs. Our findings provide insights into the molecular basis of tRNA specificity and species-specificity.     

Mechanism of the activation step of the aminoacylation reaction: a significant difference between class I and class II synthetases

In the present work we report, for the first time, a novel difference in the molecular mechanism of the activation step of aminoacylation reaction between the class I and class II aminoacyl tRNA synthetases (aaRSs). The observed difference is in the mode of nucleophilic attack by the oxygen atom of the carboxylic group of the substrate amino acid (AA) to the αP atom of adenosine triphosphate (ATP). The syn oxygen atom of the carboxylic group attacks the α-phosphorous atom (αP) of ATP in all class I aaRSs (except TrpRS) investigated, while the anti oxygen atom attacks in the case of class II aaRSs. The class I aaRSs investigated are GluRS, GlnRS, TyrRS, TrpRS, LeuRS, ValRS, IleRS, CysRS, and MetRS and class II aaRSs investigated are HisRS, LysRS, ProRS, AspRS, AsnRS, AlaRS, GlyRS, PheRS, and ThrRS. The variation of the electron density at bond critical points as a function of the conformation of the attacking oxygen atom measured by the dihedral angle ψ (C(α)-C') conclusively proves this. The result shows that the strength of the interaction of syn oxygen and αP is stronger than the interaction with the anti oxygen for class I aaRSs. This indicates that the syn oxygen is the most probable candidate for the nucleophilic attack in class I aaRSs. The result is further supported by the computation of the variation of the nonbonded interaction energies between αP atom and anti oxygen as well as syn oxygen in class I and II aaRSs, respectively. The difference in mechanism is explained based on the analysis of the electrostatic potential of the AA and ATP which shows that the relative arrangement of the ATP with respect to the AA is opposite in class I and class II aaRSs, which is correlated with the organization of the active site in respective aaRSs. A comparative study of the reaction mechanisms of the activation step in a class I aaRS (Glutaminyl tRNA synthetase) and in a class II aaRS (Histidyl tRNA synthetase) is carried out by the transition state analysis. The atoms in molecule analysis of the interaction between active site residues or ions and substrates are carried out in the reactant state and the transition state. The result shows that the observed novel difference in the mechanism is correlated with the organizations of the active sites of the respective aaRSs. The result has implication in understanding the experimentally observed different modes of tRNA binding in the two classes of aaRSs.     

Mutational separation of two pathways for editing by a class I tRNA synthetase

Aminoacyl tRNA synthetases (aaRSs) catalyze the first step in protein biosynthesis, establishing a connection between codons and amino acids. To maintain accuracy, aaRSs have evolved a second active site that eliminates noncognate amino acids. Isoleucyl tRNA synthetase edits valine by two tRNA(Ile)-dependent pathways: hydrolysis of valyl adenylate (Val-AMP, pretransfer editing) and hydrolysis of mischarged Val-tRNA(Ile) (posttransfer editing). Not understood is how a single editing site processes two distinct substrates--an adenylate and an aminoacyl tRNA ester. We report here distinct mutations within the center for editing that alter adenylate but not aminoacyl ester hydrolysis, and vice versa. These results are consistent with a molecular model that shows that the single editing active site contains two valyl binding pockets, one specific for each substrate.     

Functional defects of pathogenic human mitochondrial tRNAs related to structural fragility

Aminoacylation of transfer RNAs (tRNAs) is essential for protein synthesis. A growing number of human diseases correlate with point mutations in tRNA genes within the mitochondrial genome. These tRNAs have unique sequences that suggest they have fragile structures. However, the structural significance of pathology-related tRNA mutations and their effects on molecular function have not been explored. Here, opthalmoplegia related mutants of a human mitochondrial tRNA have been investigated. Each mutation replaces either an A-U or G-C pair in the predicted secondary structure with an A-C pair. Aminoacylation of each mutant tRNA was severely attenuated. Moreover, each strongly inhibited aminoacylation of the wild type substrate, suggesting that the effects of these mutations might not be bypassed in the potentially heteroplasmic environment of mitochondria. The function of mutant tRNAs was rescued by single compensatory mutations that restored Watson-Crick base pairing and reintroduced stability into regions of predicted secondary structure, even though the pairs introduced were different from those found in the wild type tRNA. Thus, functional defects caused by a subset of pathogenic mutations may result from the inherent structural fragility of human mitochondrial tRNAs.     

Sequence-structure-function relationships of a tRNA (m7G46) methyltransferase studied by homology modeling and site-directed mutagenesis

The Escherichia coli TrmB protein and its Saccharomyces cerevisiae ortholog Trm8p catalyze the S-adenosyl-L-methionine-dependent formation of 7-methylguanosine at position 46 (m7G46) in tRNA. To learn more about the sequence-structure-function relationships of these enzymes we carried out a thorough bioinformatics analysis of the tRNA:m7G methyltransferase (MTase) family to predict sequence regions and individual amino acid residues that may be important for the interactions between the MTase and the tRNA substrate, in particular the target guanosine 46. We used site-directed mutagenesis to construct a series of alanine substitutions and tested the activity of the mutants to elucidate the catalytic and tRNA-recognition mechanism of TrmB. The functional analysis of the mutants, together with the homology model of the TrmB structure and the results of the phylogenetic analysis, revealed the crucial residues for the formation of the substrate-binding site and the catalytic center in tRNA:m7G MTases.     

Mitochondrial translation in absence of local tRNA aminoacylation and methionyl tRNAMet formylation in Apicomplexa

Apicomplexans possess three translationally active compartments: the cytosol, a single tubular mitochondrion, and a vestigial plastid organelle called apicoplast. Mitochondrion and apicoplast are of bacterial evolutionary origin and therefore depend on a bacterial‐like translation machinery. The minimal mitochondrial genome contains only three ORFs, and in Toxoplasma gondii the absence of mitochondrial tRNA genes is compensated for by the import of cytosolic eukaryotic tRNAs. Although all compartments require a complete set of charged tRNAs, the apicomplexan nuclear genomes do not hold sufficient aminoacyl‐tRNA synthetase (aaRSs) genes to be targeted individually to each compartment. This study reveals that aaRSs are either cytosolic, apicoplastic or shared between the two compartments by dual targeting but are absent from the mitochondrion. Consequently, tRNAs are very likely imported in their aminoacylated form. Furthermore, the unexpected absence of tRNA^Met formyltransferase and peptide deformylase implies that the requirement for a specialized formylmethionyl‐tRNA^Met for translation initiation is bypassed in the mitochondrion of Apicomplexa.     

Kinetic and structural changes in HsmtPheRS,induced by pathogenic mutations in human FARS2

Mutations in the mitochondrial aminoacyl‐tRNA synthetases (mtaaRSs) can cause profound clinical presentations, and have manifested as diseases with very selective tissue specificity. To date most of the mtaaRS mutations could be phenotypically recognized, such that clinicians could identify the affected mtaaRS from the symptoms alone. Among the recently reported pathogenic variants are point mutations in FARS2 gene, encoding the human mitochondrial PheRS. Patient symptoms range from spastic paraplegia to fatal infantile Alpers encephalopathy. How clinical manifestations of these mutations relate to the changes in three‐dimensional structures and kinetic characteristics remains unclear, although impaired aminoacylation has been proposed as possible etiology of diseases. Here, we report four crystal structures of HsmtPheRS mutants, and extensive MD simulations for wild‐type and nine mutants to reveal the structural changes on dynamic trajectories of HsmtPheRS. Using steady‐state kinetic measurements of phenylalanine activation and tRNA^Phe aminoacylation, we gained insight into the structural and kinetic effects of mitochondrial disease‐related mutations in FARS2 gene.     

The hypervariable HIV-1 capsid protein residues comprise HLA-driven CD8+ T-cell escape mutations and covarying HLA-independent polymorphisms

One proposed HIV vaccine strategy is to induce Gag-specific CD8(+) T-cell responses that can corner the virus, through fitness cost of viral escape and unavailability of compensatory mutations. We show here that the most variable capsid residues principally comprise escape mutants driven by protective alleles HLA-B*57, -5801, and -8101 and covarying HLA-independent polymorphisms that arise in conjunction with these escape mutations. These covarying polymorphisms are potentially compensatory and are concentrated around three tropism-determining loops of p24, suggesting structural interdependencies. Our results demonstrate complex patterns of adaptation of HIV under immune selection pressure, the understanding of which should aid vaccine design.