FIG 2 - uploaded by Ryuya Fukunaga
Content may be subject to copyright.
Valine-binding mode. A, F o F c omit map electron densities for valine, contoured at 4.0 . Although the electron density for valine in the full-length IleRS was not resolved well, this study yielded a quite sharp electron density and we could determine the valine coordinates unambiguously. B, ball and stick representation of the active site residues, valine, and water molecules bound at the post-transfer editing site. Ionic bonds and hydrogen bonds recognizing valine are shown by dashed yellow lines (stereo view). C, the CP1 domain editing active site, rotated by 90° (stereo view)
Source publication
Isoleucyl-tRNA synthetase (IleRS) links tRNAIle with not only its cognate isoleucine but also the nearly cognate valine. The CP1 domain of IleRS deacylates, or edits, the
mischarged Val-tRNAIle. We determined the crystal structures of the Thermus thermophilus IleRS CP1 domain alone, and in its complex with valine at 1.8- and 2.0-Å resolutions, resp...
Contexts in source publication
Context 1
... Mode-The editing active site is formed mainly by one -strand (8) and two almost parallel -helices (1 and 5). In the F o F c omit map of the complex structure, there is a strong and clear electron density that could be attributed to the valine molecule in the editing active site ( Fig. 2A), and there are some electron densities that could be attributed to water molecules around the valine. The high resolution complex structure revealed the precise valine recognition mechanism (Fig. 2, B and C, and Fig. 4A (Fig. 6A). We previously proposed a valine-binding mode in the post-transfer editing state, which was modeled on the ...
Context 2
... of the complex structure, there is a strong and clear electron density that could be attributed to the valine molecule in the editing active site ( Fig. 2A), and there are some electron densities that could be attributed to water molecules around the valine. The high resolution complex structure revealed the precise valine recognition mechanism (Fig. 2, B and C, and Fig. 4A (Fig. 6A). We previously proposed a valine-binding mode in the post-transfer editing state, which was modeled on the basis of the electron density corresponding to valine (although it was not clear enough to locate it unambiguously) in the full-length IleRS structure, and the 3-terminal adenosine of tRNA Val in ...
Context 3
... does not (Fig. 5). Furthermore, the water molecule, which hydrogen bonds to Asp 328 and the NH 3 group of the substrate, is favorably located to act as the catalytic nucleophile for the ester bond hydrolysis (Fig. 6C). Conversely, in the determined valine-bound structure, Thr 230 hydrogen bonds to the valine COO group through the water molecule (Fig. 2, A and B), but the water molecule clashes with the ribose 3 of the modeled substrate, and cannot exist in the same place in the model (data not shown). We will discuss the importance of Thr 228 and Thr 230 below. In the aminoacylation reaction by IleRS, valine is first mischarged to the 2-OH of the tRNA 3 adenosine ribose (13). Under ...
Context 4
... the 3-OH valylated model, Thr 229 hydrogen bonds directly to the adenosine 5-O, as in the 2-OH valylated model, and Thr 228 and Thr 230 hydrogen bond to the adenosine 3-O through the water molecule (Fig. 7), as observed in the determined valine-bound structure (Fig. 2, A and B). This water molecule, which hydrogen bonds to Thr 228 and Thr 230 , is favorably located to act as a catalytic nucleophile for the ester bond hydrolysis (Fig. 7). In this model, Thr 228 and Thr 230 participate in the substrate recognition, in contrast to the 2-OH valylated model, thus confirming the mutational analysis that revealed ...
Similar publications
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 isoleucyltRNA synthetase (IleRS), resulting in a block to protein synthesis. Previous studies on Thermus the...
Aminoacyl-tRNA synthetases play a central role in maintaining accuracy during the translation of the genetic code. To achieve this challenging task they have to discriminate against amino acids that are very closely related not only in structure but also in chemical nature. A 'double-sieve' editing model was proposed in the late seventies to explai...
Candida albicans biofilm formation is governed by a regulatory circuit comprising nine transcription factors which control a network of target genes. However, there are still unknown genes contributing to biofilm features. Thus, the GRACE library was screened to identify genes involved in mature biofilm development. Twenty-nine conditional mutants...
Isoleucyl‐tRNA synthetase 2 (IARS2) encodes mitochondrial isoleucine‐tRNA synthetase. Pathogenic variants in the IARS2 gene are associated with mitochondrial disease. We report a female with IARS2 compound heterozygous variants, p.Val499Glyfs*14 and p.Arg784Trp who presented with infantile spasms, Leigh disease and Wolff‐Parkinson White (WPW) patte...
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 tRNA Ile binding site...
Citations
... The first site is located within the CP1 editing site (S288, W395, V500, and C502). The editing site mutations are proximal to the conserved editing catalytic aspartate (D510 based on homology) (38). The second site of mutations (E180 and N269) is located at an allosteric site near the Znf/hinge region that bridges the Rossman fold to the CP1 domain (Fig. 4A). ...
Development of antimalarial compounds into clinical candidates remains costly and arduous without detailed knowledge of the target. As resistance increases and treatment options at various stages of disease are limited, it is critical to identify multistage drug targets that are readily interrogated in biochemical assays. Whole-genome sequencing of 18 parasite clones evolved using thienopyrimidine compounds with submicromolar, rapid-killing, pan-life cycle antiparasitic activity showed that all had acquired mutations in the P. falciparum cytoplasmic isoleucyl tRNA synthetase (cIRS). Engineering two of the mutations into drug-naïve parasites recapitulated the resistance phenotype, and parasites with conditional knockdowns of cIRS became hypersensitive to two thienopyrimidines. Purified recombinant P. vivax cIRS inhibition, cross-resistance, and biochemical assays indicated a noncompetitive, allosteric binding site that is distinct from that of known cIRS inhibitors mupirocin and reveromycin A. Our data show that Plasmodium cIRS is an important chemically and genetically validated target for next-generation medicines for malaria.
... To ensure the translational fidelity, some aaRSs possess editing functions to correct such errors [53][54][55][56][57][58][59][60]. Correspondingly, two types of editing reactions are known, that is, pre-and posttransfer editing reactions, which hydrolyze a misactivated amino acid and misaminoacylated tRNA, respectively. ...
... To correct these errors, the aaRSs possess an editing function to hydrolyze the mis-products. [6][7][8][9][10][11][12][13] Two types of editing have been reported so far: hydrolysis of a mis-activated amino acid, referred to as pretransfer editing, and hydrolysis of a mis-aminoacylated tRNA, which is post-transfer editing. ...
... In E coli LRS, T247 corresponding to human LRS T293 can function in binding to isoleucine 29 along with T252, another amino acid that is conserved in the LRS editing domain among species. Both T247 and T252 are also conserved among other tRNA synthetases for bulky hydrophobic amino acids such as isoleucyl-tRNA synthetase (IRS) and valine-tRNA synthetase (VRS) in addition to LRS. 40 However, a mutation study of Thermus thermophilus IRS has revealed that T228 of IRS (which corresponds to T247 of E coli LRS) is more critical for the editing function of IRS than T233 of IRS (which corresponds to T252 of E coli LRS) when each of them is mutated to alanine, 40 suggesting that E coli LRS T247 (which corresponds to T293 of human LRS) is critical for binding to isoleucine. Although extending the study of T thermophilus IRS to human LRS might be misdirected, the study could, along with the present findings, be critical in predicting the physiological meaning of T293 LRS phosphorylation because of their conserved amino acid sequences. ...
... In E coli LRS, T247 corresponding to human LRS T293 can function in binding to isoleucine 29 along with T252, another amino acid that is conserved in the LRS editing domain among species. Both T247 and T252 are also conserved among other tRNA synthetases for bulky hydrophobic amino acids such as isoleucyl-tRNA synthetase (IRS) and valine-tRNA synthetase (VRS) in addition to LRS. 40 However, a mutation study of Thermus thermophilus IRS has revealed that T228 of IRS (which corresponds to T247 of E coli LRS) is more critical for the editing function of IRS than T233 of IRS (which corresponds to T252 of E coli LRS) when each of them is mutated to alanine, 40 suggesting that E coli LRS T247 (which corresponds to T293 of human LRS) is critical for binding to isoleucine. Although extending the study of T thermophilus IRS to human LRS might be misdirected, the study could, along with the present findings, be critical in predicting the physiological meaning of T293 LRS phosphorylation because of their conserved amino acid sequences. ...
Leucine‐rich repeat kinase 2 (LRRK2) is a causal gene of Parkinson disease. G2019S pathogenic mutation increases its kinase activity. LRRK2 regulates various phenotypes including autophagy, neurite outgrowth, and vesicle trafficking. Leucyl‐tRNA synthetase (LRS) attaches leucine to tRNALeu and activates mTORC1. Down‐regulation of LRS induces autophagy. We investigated the relationship between LRRK2 and LRS in regulating autophagy and observed interaction between endogenous LRRK2 and LRS proteins and LRS phosphorylation by LRRK2. Mutation studies implicated that T293 in the LRS editing domain was a putative phosphorylation site. Phospho‐Thr in LRS was increased in cells overexpressing G2019S and dopaminergic neurons differentiated from induced pluripotent stem (iPS) cells of a G2019S carrier. It was decreased by treatment with an LRRK2 kinase inhibitor (GSK2578215A). Phosphomimetic T293D displayed lower leucine bindings than wild type (WT), suggesting its defective editing function. Cellular expression of T293D increased expression of GRP78/BiP, LC3B‐II, and p62 proteins and number of LC3 puncta. Increase of GRP78 and phosphorylated LRS was diminished by treatment with GSK2578215A. Levels of LC3B, GRP78/BiP, p62, and α‐synuclein proteins were also increased in G2019S transgenic (TG) mice. These data suggest that LRRK2‐mediated LRS phosphorylation impairs autophagy by increasing protein misfolding and endoplasmic reticulum stress mediated by LRS editing defect.
Significance of the study
Leucine‐rich repeat kinase 2 (LRRK2) is the most common genetic cause of Parkinson disease (PD), and the most prevalent pathogenic mutation, G2019S, increases its kinase activity. In this study, we elucidated that leucyl‐tRNA synthetase (LRS) was an LRRK2 kinase substrate and identified T293 as an LRRK2 phosphorylation site. LRRK2‐meidated LRS phosphorylation or G2019S can lead to impairment of LRS editing, increased ER stress, and accumulation of autophagy markers. These results demonstrate that LRRK2 kinase activity can facilitate accumulation of misfolded protein, suggesting that LRRK2 kinase might be a potential PD therapeutic target along with previous studies.
... To correct such errors, these aaRSs possess an editing function that hydrolyzes mis-products. [4][5][6][7][8][9][10][11] Two types of editing have been reported so far; hydrolysis of a mis-activated amino acid is referred to as pre-transfer editing, and that of mis-aminoacylated tRNA as post-transfer editing. The active site of editing is located in the connective polypeptide 1 (CP1) domain, whereas that of aminoacylation is located in the Rossmann-fold catalytic domain. ...
We analyzed the electronic structural changes that occur in the reaction cycle of a biological catalyst composed of RNA and protein, and elucidated the dynamical rearrangements of the electronic structure that was obtained from our previous study in which ab initio quantum mechanics=molecular mechanics molecular dynamics simulations were performed. Notable results that we obtained include the generation of a reactive HOMO that is responsible for bond formation in the initial stages of the reaction, and the appearance of a reactive LUMO that is involved in the bond rupture that leads to products. We denote these changes as dynamical induction of the reactive HOMO (DIRH) and LUMO (DIRL), respectively. Interestingly, we also find that the induction of the reactive HOMO is enhanced by the formation of a low-barrier hydrogen bond (LBHB), which, to the best of our knowledge, represents a novel role for LBHBs in enzymatic systems.
... Cell expressing IleRS(T233P) are defective for quality control of mischarged tRNA Ile . IleRS was chosen for this study as it has a well characterized post-transfer editing activity, and substitutions that disrupt this editing without affecting aminoacylation have been made 10,11,23 . While several studies have looked at the role of QC by IleRS in E. coli, relatively few studies have examined the importance of QC in other organisms. ...
... The point mutation introduced causes a substitution of Thr at codon 233 to Pro. This T233P substitution was chosen because a similar substitution in IleRS of Escherichia coli was shown to disrupt QC by this enzyme, and while other substitutions in E. coli IleRS disrupt QC, the T233P substitution has virtually no effect on aminoacylation activity 14,23 . To test whether the cells with the ileS(T233P) allele lacked tRNA Ile QC activity, cell lysates of the wild-type ileS and mutant ileS(T233P) were tested for their ability to charge tRNA Ile with either the cognate Ile or the non-cognate amino acids, Val and Leu. ...
Isoleucyl-tRNA synthetase (IleRS) is an aminoacyl-tRNA synthetase whose essential function is to aminoacylate tRNAIle with isoleucine. Like some other aminoacyl-tRNA synthetases, IleRS can mischarge tRNAIle and correct this misacylation through a separate post-transfer editing function. To explore the biological significance of this editing function, we created a ileS(T233P) mutant of Bacillus subtilis that allows tRNAIle mischarging while retaining wild-type Ile-tRNAIle synthesis activity. As seen in other species defective for aminoacylation quality control, the growth rate of the ileS(T233P) strain was not significantly different from wild-type. When the ileS(T233P) strain was assessed for its ability to promote distinct phenotypes in response to starvation, the ileS(T233P) strain was observed to exhibit a significant defect in formation of environmentally resistant spores. The sporulation defect ranged from 3-fold to 30-fold and was due to a delay in activation of early sporulation genes. The loss of aminoacylation quality control in the ileS(T233P) strain resulted in the inability to compete with a wild-type strain under selective conditions that required sporulation. These data show that the quality control function of IleRS is required in B. subtilis for efficient sporulation and suggests that editing by aminoacyl-tRNA synthetases may be important for survival under starvation/nutrient limitation conditions.
... 22,23 In contrast, post-transfer editing typically utilizes a second active site, the editing site, whose role is to remove the aminoacyl moiety from misacylated tRNA. 13,21,24 For example, threonyl-tRNA synthetase is thought to discern among threonine, serine, and valine 9,13,14 via a double-sieve model. 25,26 First, its aminoacylation site acts to exclude valine, while its editing site catalyzes the hydrolytic removal of serinyl from misacylated tRNA Thr . ...
Aminoacyl-tRNA synthetases (aaRSs) are central to a number of physiological processes including protein biosynthesis. In particular, they activate and then transfer their corresponding amino acid to the cognate tRNA. This is achieved with a generally remarkably high fidelity by editing against incorrect standard and non-standard amino acids. Using Docking, molecular dynamics (MD) and hybrid quantum mechanical/molecular mechanics methods, we have investigated mechanisms by which methionyl-tRNA synthetase (MetRS) may edit against the highly toxic, non-cognate, amino acids homocysteine (Hcy) and its oxygen analogue homoserine (Hse). Substrate-assisted editing of Hcy-AMP in which its own phosphate acts as the mechanistic base occurs with a rate limiting barrier of 98.2 kJ mol-1. This step corresponds to nucleophilic attack of the Hcy side-chain sulfur at its own carbonyl carbon (CCarb). In contrast, a new possible editing mechanism is identified in which an active site aspartate (Asp259) acts as the base. The rate-limiting step is now rotation about the substrates aminoacyl Cβ-Cγ bond with a barrier of 27.5 kJ mol-1 while for Hse-AMP the rate limiting step is cleavage of the CCarb-OP bond with a barrier of 30.9 kJ mol-1. A similarly positioned aspartate or glutamate also occurs in the homologous enzymes LeuRS, IleRS and ValRS, which also discriminate against Hcy. Docking and MD studies suggest that at least in the case of LeuRS and ValRS, a similar editing mechanism may be possible.
... A number of aaRSs, which catalyze the aminoacylation of branched chain amino acids, such as LeuRS, valyl-tRNA synthetase (ValRS) and IleRS, possess an additional proofreading domain called the CP1 domain [77][78][79]. This domain is located about ~30 Å away from the aminoacylation active site and is involved in recognizing and hydrolyzing misacylated amino acids on the 3'-end of the tRNA Leu . ...
Transfer RNAs (tRNAs) are central players in the protein translation machinery and as such are prominent targets for a large number of natural and synthetic antibiotics. This review focuses on the role of tRNAs in bacterial antibiosis. We will discuss examples of antibiotics that target multiple stages in tRNA biology from tRNA biogenesis and modification, mature tRNAs, aminoacylation of tRNA as well as prevention of proper tRNA function by small molecules binding to the ribosome. Finally, the role of deacylated tRNAs in the bacterial "stringent response" mechanism that can lead to bacteria displaying antibiotic persistence phenotypes will be discussed.
... The structural domains of a protein have often been identified through the use of limited proteolysis [5][6][7][8], since the connections between protein domains are exposed and thus are better protease substrates. Therefore, limited proteolysis has been widely used to isolate domains, not only to identity function [9], but also to create smaller units for structural analysis by X-ray crystallography or, more increasingly, by NMR spectrometry [10]. Moreover, it can also provide effective information for structural and functional analyses of proteins. ...
The structural domains of proteins have often been identified through the use of limited proteolysis. In structural genomics studies, it is necessary to carry this out in a high-throughput manner. Here, we constructed a novel high-throughput system, which consists of cell-free protein expression and one-step affinity purification, followed by limited proteolysis using a unique new method, referred to "on beads method". All these steps were carried out on 96-well plate formats and completed in two days, even by manual handling. The merits of the new method versus the conventional one are as follows: (1) experimental times are reduced, (2) the sample preparation for limited proteolysis experiments is simplified, and (3) both protein purification and limited digestion can be performed "in situ" on the same sample plate. This preparation method is therefore suitable for highly automated, proteolytic analyses coupled to mass spectrometry techniques at a micro-scale protein expression level. The resulting protease-resistant fragments were analyzed by MALDI-TOF-MS and protein domains of 34 mouse cDNA products were identified with this system.
... (i) LeuRS/IleRS/ValRS systems. Extensive studies have revealed that the class I LeuRS, IleRS and ValRS share a homologous CP1 domain for post-transfer editing [47,[61][62][63][64][65][66]. The CP1 domain is inserted into the Rossmann fold aminoacylation domain and separates it into two halves [54]. ...
... Manipulation of the pocket size by introducing a larger or a smaller residue at this site could artificially shift the editing specificity of post-transfer editing. A very similar mechanism is also used in the closely related IleRS and ValRS systems [63,64]. From the crystal structure, the editing active site of the N2 domain of E. coli ThrRS (EcThrRS) is just suitable for binding Ser-tRNA Thr . ...
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.
