Fidelity of translation, which is predominately dictated by the accuracy of aminoacyl-tRNA synthetases in pairing amino acids with correct tRNAs, is of central importance in biology. Yet, deliberate modifications of translational fidelity can be beneficial. Here we found human and not E. coli AlaRS has an intrinsic capacity for mispairing alanine onto nonalanyl-tRNAs including tRNACys. Consistently, a cysteine-to-alanine substitution was found in a reporter protein expressed in human cells. All human AlaRS-mischarged tRNAs have a G4:U69 base pair in the acceptor stem. The base pair is required for the mischarging. By solving the crystal structure of human AlaRS and comparing it to that of E. coli AlaRS, we identified a key sequence divergence between eukaryotes and bacteria that influences mischarging. Thus, the expanded tRNA specificity of AlaRS appears to be an evolutionary gain-of-function to provide posttranscriptional alanine substitutions in eukaryotic proteins for potential regulations.

Over the past decade, aminoacyl-tRNA synthetases (AARSs) have emerged as a new class of regulatory proteins with widespread functions beyond their classic role in protein synthesis. The functional expansion concurs with the incorporation of new domains and motifs to AARSs and coincides with the emergence of the multi-synthetase complex (MSC) during the course of eukaryotic evolution. Notably, the new domains in AARSs are often found to be structurally disordered or to be linked to the enzyme cores via unstructured linkers. We performed bioinformatic analysis and classified the 20 human cytoplasmic AARSs into three groups based on their propensities for structural disorder. The analysis also suggests that, while the assembly of the MSC mainly involves ordered structural domains, structurally disordered regions play an important role in activating and expanding the regulatory functions of AARSs.

The question of how dispersed mutations in one protein engender the same gain-of-function phenotype is of great interest. Here we focus on mutations in glycyl-tRNA synthetase (GlyRS) that cause an axonal form of Charcot–Marie–Tooth (CMT) diseases, the most common hereditary peripheral neuropathies. Because the disease phenotype is dominant, and not correlated with defects in the role of GlyRS in protein synthesis, the mutant proteins are considered to be neomorphs that gain new functions from altered protein structure. Given that previous crystal structures showed little conformational difference between dimeric wild-type and CMT-causing mutant GlyRSs, the mutant proteins were investigated in solution by hydrogen-deuterium exchange (monitored by mass spectrometry) and small-angle X-ray scattering to uncover structural changes that could be suppressed by crystal packing interactions. Significantly, each of five spatially dispersed mutations induced the same conformational opening of a consensus area that is mostly buried in the wild-type protein. The identified neomorphic surface is thus a candidate for making CMT-associated pathological interactions, and a target for disease correction. Additional result showed that a helix-turn-helix WHEP domain that was appended to GlyRS in metazoans can regulate the neomorphic structural change, and that the gain of function of the CMT mutants might be due to the loss of function of the WHEP domain as a regulator. Overall, the results demonstrate how spatially dispersed and seemingly unrelated mutations can perpetrate the same localized effect on a protein.

Human lysyl-tRNA synthetase is bound to the multi-tRNA synthetase complex (MSC) that maintains and regulates the aminoacylation and nuclear functions of LysRS. The p38 scaffold protein binds LysRS to the MSC and, only with the appropriate cue, mobilizes LysRS for redirection to the nucleus to interact with the microphthalmia associated transcription factor (MITF). In recent work, an (α2)2 LysRS tetramer crystallized to yield a high-resolution structure and raised the question of how LysRS is arranged (dimer or tetramer) in the MSC to interact with p38. To understand the structural organization of the LysRS-p38 complex that regulates LysRS mobilization, we investigated the complex by use of small angle X-ray scattering and hydrogen-deuterium exchange with mass spectrometry in solution. The structure revealed a surprising α2β1∶β1α2 organization in which a dimeric p38 scaffold holds two LysRS α2 dimers in a parallel configuration. Each of the N-terminal 48 residues of p38 binds one LysRS dimer and, in so doing, brings two copies of the LysRS dimer into the MSC. The results suggest that this unique geometry, which reconfigures the LysRS tetramer from α2∶α2 to α2β1∶β1α2, is designed to control both retention and mobilization of LysRS from the MSC.

Aminoacyl tRNA synthetases are known for catalysis of aminoacylation. Significantly, some mammalian synthetases developed cytokine functions possibly linked to disease-causing mutations in tRNA synthetases. Not understood is how epitopes for cytokine signaling were introduced into catalytic scaffolds without disturbing aminoacylation. Here we investigate human tyrosyl-tRNA synthetase, where a catalytic-domain surface helix, next to the active site, was recruited for interleukin-8-like cytokine signaling. Taking advantage of our high resolution structure, the reciprocal impact of rational mutations designed to disrupt aminoacylation or cytokine signaling was investigated with multiple assays. The collective analysis demonstrated a protective fine-structure separation of aminoacylation from cytokine activities within the conserved catalytic domain. As a consequence, disease-causing mutations affecting cell signaling can arise without disturbing aminoacylation. These results with TyrRS also predict the previously unknown binding conformation of interleukin-8-like CXC cytokines.

In mammals, many aminoacyl-tRNA synthetases are bound together in a multisynthetase complex (MSC) as a reservoir of procytokines and regulation molecules for functions beyond aminoacylation. The α2 homodimeric lysyl-tRNA synthetase (LysRS) is tightly bound in the MSC and, under specific conditions, is secreted to trigger a proinflammatory response. Results by others suggest that α2 LysRS is tightly bound into the core of the MSC with homodimeric β2 p38, a scaffolding protein that itself is multifunctional. Not understood is how the two dimeric proteins combine to make a presumptive α2β2 heterotetramer and, in particular, the location of the surfaces on LysRS that would accommodate the p38 interactions. Here we present a 2.3-Å crystal structure of a tetrameric form of human LysRS. The relatively loose (as seen in solution) tetramer interface is assembled from two eukaryote-specific sequences, one in the catalytic- and another in the anticodon-binding domain. This same interface is predicted to provide unique determinants for interaction with p38. The analyses suggest how the core of the MSC is assembled and, more generally, that interactions and functions of synthetases can be built and regulated through dynamic protein–protein interfaces. These interfaces are created from small adaptations to what is otherwise a highly conserved (through evolution) polypeptide sequence.

Disease-causing mutations occur in genes for aminoacyl tRNA synthetases. That some mutations are dominant suggests a gain of function. Native tRNA synthetases, such as tyrosyl-tRNA synthetase (TyrRS) and tryptophanyl-tRNA synthetase, catalyze aminoacylation and are also procytokines that are activated by natural fragmentation. In principle, however, gain-of-function phenotypes could arise from mutational activation of synthetase procytokines. From crystal structure analysis, we hypothesized that a steric block of a critical Glu-Leu-Arg (ELR) motif in full-length TyrRS suppresses the cytokine activity of a natural fragment. To test this hypothesis, we attempted to uncover ELR in the procytokine by mutating a conserved tyrosine (Y341) that tethers ELR. Site-specific proteolytic cleavage and small-angle X-ray scattering established subtle opening of the structure by the mutation. Strikingly, four different assays demonstrated mutational activation of cytokine functions. The results prove the possibilities for constitutive gain-of-function mutations in tRNA synthetases.

Functional expansion of specific tRNA synthetases in higher organisms is well documented. These additional functions may explain why dominant mutations in glycyl-tRNA synthetase (GlyRS) and tyrosyl-tRNA synthetase cause Charcot–Marie–Tooth (CMT) disease, the most common heritable disease of the peripheral nervous system. At least 10 disease-causing mutant alleles of GlyRS have been annotated. These mutations scatter broadly across the primary sequence and have no apparent unifying connection. Here we report the structure of wild type and a CMT-causing mutant (G526R) of homodimeric human GlyRS. The mutation is at the site for synthesis of glycyl-adenylate, but the rest of the two structures are closely similar. Significantly, the mutant form diffracts to a higher resolution and has a greater dimer interface. The extra dimer interactions are located ≈30 Å away from the G526R mutation. Direct experiments confirm the tighter dimer interaction of the G526R protein. The results suggest the possible importance of subtle, long-range structural effects of CMT-causing mutations at the dimer interface. From analysis of a third crystal, an appended motif, found in higher eukaryote GlyRSs, seems not to have a role in these long-range effects.

Charcot–Marie–Tooth (CMT) diseases are the most common heritable peripheral neuropathy. At least 10 different mutant alleles of GARS (the gene for glycyl-tRNA synthetase) have been reported to cause a dominant axonal form of CMT (type 2D). A unifying connection between these mutations and CMT has been unclear. Here, mapping mutations onto the recently determined crystal structure of human GlyRS showed them within a band encompassing both sides of the dimer interface, with two CMT-causing mutations being at sites that are complementary partners of a “kissing” contact across the dimer interface. The CMT phenotype is shown here to not correlate with aminoacylation activity. However, most mutations affect dimer formation (to enhance or weaken). Seven CMT-causing variants and the wild-type protein were expressed in transfected neuroblastoma cells that sprout primitive neurites. Wild-type GlyRS distributed into the nascent neurites and was associated with normal neurite sprouting. In contrast, all mutant proteins were distribution-defective. Thus, CMT-causing mutations of GlyRS share a common defect in localization. This defect may be connected in some way to a change in the surfaces at the dimer interface.

•Crystal structure of human SerRS in complex with Ser-SA solved at 2.9 Å resolution•Ser-SA binding induces conformational change of SerRS specific to higher eukaryotes•Ser-SA binding stabilizes conformation of higher eukaryote-specific insertions•Long-range structural and functional communication between active site and insertion ISeryl-tRNA synthetase (SerRS), an essential enzyme for translation, also regulates vascular development. This “gain-of-function” has been linked to the UNE-S domain added to vertebrate SerRS during evolution. However, the significance of two insertions also specific to higher eukaryotic SerRS remains elusive. Here, we determined the crystal structure of human SerRS in complex with Ser-SA, an aminoacylation reaction intermediate analog, at 2.9 Å resolution. Despite a 70 Å distance, binding of Ser-SA in the catalytic domain dramatically leverages the position of Insertion I in the tRNA binding domain. Importantly, this leverage is specific to higher eukaryotes and not seen in bacterial, archaeal, and lower eukaryotic SerRSs. Deletion of Insertion I does not affect tRNA binding but instead reduce the catalytic efficiency of the synthetase. Thus, a long-range conformational and functional communication specific to higher eukaryotes is found in human SerRS, possibly to coordinate translation with vasculogenesis.Download high-res image (197KB)Download full-size image

•Angiogenin and oxidative stress trigger TyrRS nuclear translocation from cytosol•Nuclear TyrRS promotes expression of DNA damage response genes by activating E2F1•TyrRS interacts with TRIM28 and selectively blocks HDAC1 activity•TyrRS functions to protect cell and organism from DNA damageTyrosyl-tRNA synthetase (TyrRS) is known for its essential aminoacylation function in protein synthesis. Here we report a function for TyrRS in DNA damage protection. We found that oxidative stress, which often downregulates protein synthesis, induces TyrRS to rapidly translocate from the cytosol to the nucleus. We also found that angiogenin mediates or potentiates this stress-induced translocalization. The nuclear-localized TyrRS activates transcription factor E2F1 to upregulate the expression of DNA damage repair genes such as BRCA1 and RAD51. The activation is achieved through direct interaction of TyrRS with TRIM28 to sequester this vertebrate-specific epigenetic repressor and its associated HDAC1 from deacetylating and suppressing E2F1. Remarkably, overexpression of TyrRS strongly protects against UV-induced DNA double-strand breaks in zebrafish, whereas restricting TyrRS nuclear entry completely abolishes the protection. Therefore, oxidative stress triggers an essential cytoplasmic enzyme used for protein synthesis to translocate to the nucleus to protect against DNA damage.Download high-res image (364KB)Download full-size image

Known as an essential component of the translational apparatus, the aminoacyl-tRNA synthetase family catalyzes the first step reaction in protein synthesis, that is, to specifically attach each amino acid to its cognate tRNA. While preserving this essential role, tRNA synthetases developed other roles during evolution. Human tRNA synthetases, in particular, have diverse functions in different pathways involving angiogenesis, inflammation and apoptosis. The functional diversity is further illustrated in the association with various diseases through genetic mutations that do not affect aminoacylation or protein synthesis. Here we review the accumulated knowledge on how human tRNA synthetases used structural inventions to achieve functional expansions.

In this issue of Molecular Cell, Ivanov et al. (2011) provide mechanistic insight into tRNA fragment-based translational regulation to expand our understanding of the nondecoding roles of tRNA.

Higher eukaryote tRNA synthetases have expanded functions that come from enlarged, more differentiated structures that were adapted to fit aminoacylation function. How those adaptations affect catalytic mechanisms is not known. Presented here is the structure of a catalytically active natural splice variant of human tryptophanyl-tRNA synthetase (TrpRS) that is a potent angiostatic factor. This and related structures suggest that a eukaryote-specific N-terminal extension of the core enzyme changed substrate recognition by forming an active site cap. At the junction of the extension and core catalytic unit, an arginine is recruited to replace a missing landmark lysine almost 200 residues away. Mutagenesis, rapid kinetic, and substrate binding studies support the functional significance of the cap and arginine recruitment. Thus, the enzyme function of human TrpRS has switched more to the N terminus of the sequence. This switch has the effect of creating selective pressure to retain the N-terminal extension for functional expansion.

•Methods for studying nuclear function of AARS are proposed.•Cell fractionation used for determining the nuclear localization of AARS is described.•DNA-cellulose pull-down assay for analyzing the direct interaction of AARS with DNA is provided.•Chromatin immunoprecipitation assay for identifying the AARS binding loci in the genome is described in detail.Aminoacyl tRNA synthetases (AARSs) are best known for their essential role in translation in the cytoplasm. The concept that AARSs also exist in the nucleus started to draw attention around the turn of the new millennium, when aminoacylated tRNAs were first found in the nuclei of Xenopus oocytes. It is now expected that all cytoplasmic AARSs are present in the nucleus. In addition to tRNA aminoacylation, nuclear AARSs were found to regulate a spectrum of biological processes and responses, with many AARSs functioning through regulation at the level of gene transcription. In this paper, we focus on describing methods that have been successfully implemented to study AARSs in transcriptional regulation. These include a cell fractionation assay to detect nuclear localization, an in vitro DNA-cellulose pull-down assay to determine DNA binding capacity, and a chromatin immunoprecipitation (ChIP)-DNA deep sequencing assay to identify DNA binding sites. Application of these methods would expand our understanding of AARS functions and reveal critical insights on the coordination of gene transcription and translation.