Structural basis for the dynamics of human methionyl-tRNA synthetase in multi-tRNA synthetase complexes

In mammals, eight aminoacyl-tRNA synthetases (AARSs) and three AARS-interacting multifunctional proteins (AIMPs) form a multi-tRNA synthetase complex (MSC). MSC components possess extension peptides for MSC assembly and specific functions. Human cytosolic methionyl-tRNA synthetase (MRS) has appended peptides at both termini of the catalytic main body. The N-terminal extension includes a glutathione transferase (GST) domain responsible for interacting with AIMP3, and a long linker peptide between the GST and catalytic domains. Herein, we determined crystal structures of the human MRS catalytic main body, and the complex of the GST domain and AIMP3. The structures reveal human-specific structural details of the MRS, and provide a dynamic model for MRS at the level of domain orientation. A movement of zinc knuckles inserted in the catalytic domain is required for MRS catalytic activity. Depending on the position of the GST domain relative to the catalytic main body, MRS can either block or present its tRNA binding site. Since MRS is part of a huge MSC, we propose a dynamic switching between two possible MRS conformations; a closed conformation in which the catalytic domain is compactly attached to the MSC, and an open conformation with a free catalytic domain dissociated from other MSC components.     

Reinvestigation of Aminoacyl-TRNA Synthetase Core Complex by Affinity Purification-Mass Spectrometry Reveals TARSL2 as a Potential Member of the Complex

Twenty different aminoacyl-tRNA synthetases (ARSs) link each amino acid to their cognate tRNAs. Individual ARSs are also associated with various non-canonical activities involved in neuronal diseases, cancer and autoimmune diseases. Among them, eight ARSs (D, EP, I, K, L, M, Q and RARS), together with three ARS-interacting multifunctional proteins (AIMPs), are currently known to assemble the multi-synthetase complex (MSC). However, the cellular function and global topology of MSC remain unclear. In order to understand the complex interaction within MSC, we conducted affinity purification-mass spectrometry (AP-MS) using each of AIMP1, AIMP2 and KARS as a bait protein. Mass spectrometric data were funneled into SAINT software to distinguish true interactions from background contaminants. A total of 40, 134, 101 proteins in each bait scored over 0.9 of SAINT probability in HEK 293T cells. Complex-forming ARSs, such as DARS, EPRS, IARS, Kars, LARS, MARS, QARS and RARS, were constantly found to interact with each bait. Variants such as, AIMP2-DX2 and AIMP1 isoform 2 were found with specific peptides in KARS precipitates. Relative enrichment analysis of the mass spectrometric data demonstrated that TARSL2 (threonyl-tRNA synthetase like-2) was highly enriched with the ARS-core complex. The interaction was further confirmed by coimmunoprecipitation of TARSL2 with other ARS core-complex components. We suggest TARSL2 as a new component of ARS core-complex.     

Discovery of two distinct aminoacyl-tRNA synthetase complexes anchored to the Plasmodium surface tRNA import protein

Aminoacyl-tRNA synthetases (aaRSs) attach amino acids to their cognate transfer RNAs. In eukaryotes, a subset of cytosolic aaRSs is organized into a multisynthetase complex (MSC), along with specialized scaffolding proteins referred to as aaRS-interacting multifunctional proteins (AIMPs). In Plasmodium, the causative agent of malaria, the tRNA import protein (tRip), is a membrane protein that participates in tRNA trafficking; we show that tRip also functions as an AIMP. We identified three aaRSs, the glutamyl-tRNA synthetase (ERS), glutaminyl-tRNA synthetase (QRS), and methionyl-tRNA synthetase (MRS), which were specifically coimmunoprecipitated with tRip in Plasmodium berghei blood stage parasites. All four proteins contain an N-terminal glutathione-S-transferase (GST)–like domain that was demonstrated to be involved in MSC assembly. In contrast to previous studies, further dissection of GST-like interactions identified two exclusive heterotrimeric complexes: the Q-complex (tRip–ERS–QRS) and the M-complex (tRip–ERS–MRS). Gel filtration and light scattering suggest a 2:2:2 stoichiometry for both complexes but with distinct biophysical properties and mutational analysis further revealed that the GST-like domains of QRS and MRS use different strategies to bind ERS. Taken together, our results demonstrate that neither the singular homodimerization of tRip nor its localization in the parasite plasma membrane prevents the formation of MSCs in Plasmodium. Besides, the extracellular localization of the tRNA-binding module of tRip is compensated by the presence of additional tRNA-binding modules fused to MRS and QRS, providing each MSC with two spatially distinct functions: aminoacylation of intraparasitic tRNAs and binding of extracellular tRNAs. This unique host–pathogen interaction is discussed.     

AIMP3/p18 Controls Translational Initiation by Mediating the Delivery of Charged Initiator tRNA to Initiation Complex

Aminoacyl-tRNA synthetase-interacting multifunctional proteins (AIMPs) are nonenzymatic scaffolding proteins that comprise multisynthetase complex (MSC) with nine aminoacyl-tRNA synthetases in higher eukaryotes. Among the three AIMPs, AIMP3/p18 is strongly anchored to methionyl-tRNA synthetase (MRS) in the MSC. MRS attaches methionine (Met) to initiator tRNA (tRNA_i^Met) and plays an important role in translation initiation. It is known that AIMP3 is dispatched to nucleus or nuclear membrane to induce DNA damage response or senescence; however, the role of AIMP3 in translation as a component of MSC and the meaning of its interaction with MRS are still unclear. Herein, we observed that AIMP3 specifically interacted with Met-tRNA_i^Metin vitro, while it showed little or reduced interaction with unacylated or lysine-charged tRNA_i^Met. In addition, AIMP3 discriminates Met-tRNA_i^Met from Met-charged elongator tRNA based on filter-binding assay. Pull‐down assay revealed that AIMP3 and MRS had noncompetitive interaction with eukaryotic initiation factor 2 (eIF2) γ subunit (eIF2γ), which is in charge of binding with Met-tRNA_i^Met for the delivery of Met-tRNA_i^Met to ribosome. AIMP3 recruited active eIF2γ to the MRS-AIMP3 complex, and the level of Met-tRNA_i^Met bound to eIF2 complex was reduced by AIMP3 knockdown resulting in reduced protein synthesis. All these results suggested the novel function of AIMP3 as a critical mediator of Met-tRNA_i^Met transfer from MRS to eIF2 complex for the accurate and efficient translation initiation.     

Evaluation of Multi-tRNA Synthetase Complex by Multiple Reaction Monitoring Mass Spectrometry Coupled with Size Exclusion Chromatography

Eight aminoacyl-tRNA synthetases (M, K, Q, D, R, I, EP and LARS) and three auxiliary proteins (AIMP1, 2 and 3) are known to form a multi-tRNA synthetase complex (MSC) in mammalian cells. We combined size exclusion chromatography (SEC) with reversed-phase liquid chromatography multiple reaction monitoring mass spectrometry (RPLC-MRM-MS) to characterize MSC components and free ARS proteins in human embryonic kidney (HEK 293T) cells. Crude cell extract and affinity-purified proteins were fractionated by SEC in non-denaturing state and ARSs were monitored in each fraction by MRM-MS. The eleven MSC components appeared mostly in earlier SEC fractions demonstrating their participation in complex formation. TARSL2 and AIMP2-DX2, despite their low abundance, were co-purified with KARS and detected in the SEC fractions, where MSC appeared. Moreover, other large complex-forming ARS proteins, such as VARS and FARS, were detected in earlier fractions. The MRM-MS results were further confirmed by western blot analysis. Our study demonstrates usefulness of combined SEC-MRM analysis for the characterization of protein complexes and in understanding the behavior of minor isoforms or variant proteins.     

Aminoacyl-tRNA synthetases of the multi-tRNA synthetase complex and their role in tumorigenesis

In mammalian cells, 20 aminoacyl-tRNA synthetases (AARS) catalyze the ligation of amino acids to their cognate tRNAs to generate aminoacylated-tRNAs. In higher eukaryotes, 9 of the 20 AARSs, along with 3 auxiliary proteins, join to form the cytoplasmic multi-tRNA synthetase complex (MSC). The complex is absent in prokaryotes, but evolutionary expansion of MSC constituents, primarily by addition of novel interacting domains, facilitates formation of subcomplexes that join to establish the holo-MSC. In some cases, environmental cues direct the release of constituents from the MSC which enables the execution of non-canonical, i.e., “moonlighting”, functions distinct from their essential activities in protein translation. These activities are generally beneficial, but can also be deleterious to the cell. Elucidation of the non-canonical activities of several AARSs residing in the MSC suggest they are potential therapeutic targets for cancer, as well as metabolic and neurologic diseases. Here, we describe the role of MSC-resident AARSs in cancer progression, and the factors that regulate their release from the MSC. Also, we highlight recent developments in therapeutic modalities that target MSC AARSs for cancer prevention and treatment.     

Crystal structure of human cytosolic aspartyl‐tRNA synthetase,a component of multi‐tRNA synthetase complex

Human cytosolic aspartyl‐tRNA synthetase (DRS) catalyzes the attachment of the amino acid aspartic acid to its cognate tRNA and it is a component of the multi‐tRNA synthetase complex (MSC) which has been known to be involved in unexpected signaling pathways. Here, we report the crystal structure of DRS at a resolution of 2.25 Å. DRS is a homodimer with a dimer interface of 3750.5 Ų which comprises 16.6% of the monomeric surface area. Our structure reveals the C‐terminal end of the N‐helix which is considered as a unique addition in DRS, and its conformation further supports the switching model of the N‐helix for the transfer of tRNA^Asp to elongation factor 1α. From our analyses of the crystal structure and post‐translational modification of DRS, we suggest that the phosphorylation of Ser146 provokes the separation of DRS from the MSC and provides the binding site for an interaction partner with unforeseen functions.Proteins 2013; 81:1840–1846. © 2013 Wiley Periodicals, Inc.     

Determination of three-dimensional structure and residues of the novel tumor suppressor AIMP3/p18 required for the interaction with ATM

Although AIMP3/p18 is normally associated with the multi-tRNA synthetase complex via its specific interaction with methionyl-tRNA synthetase, it also works as a tumor suppressor by interacting with ATM, the upstream kinase of p53. To understand the molecular interactions of AIMP3 and the mechanisms involved, we determined the crystal structure of AIMP3 at 2.0-angstroms resolution and identified its potential sites of interaction with ATM. AIMP3 contains two distinct domains linked by a 7-amino acid (Lys57-Ser63) peptide, which contains a 3(10) helix. The 56-amino acid N-terminal domain consists of two helices into which three antiparallel beta strands are inserted, and the 111-amino acid C-terminal domain contains a bundle of five helices (Thr64-Tyr152) followed by a coiled region (Pro153-Leu169). Structural analyses revealed homologous proteins such as yeast glutamyl-tRNA synthetase, Arc1p, EF1Bgamma, and glutathione S-transferase and suggested two potential molecular binding sites. Moreover, mutations at the C-terminal putative binding site abolished the interaction between AIMP3 and ATM and the ability of AIMP3 to activate p53. Thus, this work identified the two potential molecular interaction sites of AIMP3 and determined the residues critical for its tumor-suppressive activity through the interaction with ATM.     

苯丙氨酰-tRNA合成酶的进化与结构域丢失

基因的复制、融合以及基因的水平转移是许多蛋白质包括氨酰 tRNA合成酶 (aminoacyl tRNAsynthetase ,AARS)进化过程中的常见事件。然而作者研究的结果显示 ,苯丙氨酰 tRNA合成酶 (phenylalanyl tRNAsynthetase,PheRS)的进化主要表现为一些结构域的丢失 ;并且这种结构域的丢失不影响PheRS的功能或活性。通常在生物从细菌到真核生物的进化过程中 ,其基因组的大小和基因的数目都有所增加 ,然而有趣的是 ,真核生物中PheRS的结构域类型和数目都明显少于细菌的PheRS。PheRS通过结构域的丢失而进化的现象 ,似乎与某些AARS功能由多重专一性向单一专一性的演化有着“异曲同工”之妙。     

Contributions of aminoacyl-tRNA synthetase-interacting multifunctional protein-3 to mammalian translation initiation

Aminoacyl-tRNA synthetase-interacting multifunctional protein-3 (AIMP3) stabilizes and protects mammalian methionyl-tRNA synthetase (MRS) and eukaryotic initiation factor 2 subunit gamma (eIF2γ), factors involved in the formation and the delivery of Met-tRNA _i ^Met respectively, through the binding interactions. Due to the protections that MRS and eIF2γ are provided from the interactions with AIMP3, cellular levels of MRS and eIF2γ may be able to be maintained high enough for their canonical and/or non-canonical functions.     

Internally Deleted Human tRNA Synthetase Suggests Evolutionary Pressure for Repurposing

Aminoacyl-tRNA synthetases (AARSs) catalyze aminoacylation of tRNAs in the cytoplasm. Surprisingly,?AARSs also have critical extracellular and nuclear functions. Evolutionary pressure for new functions might be manifested by splice variants that skip only an internal catalytic domain (CD) and link noncatalytic N- and C-terminal polypeptides. Using disease-associated histidyl-tRNA synthetase (HisRS) as an example, we found an expressed 171-amino acid protein (HisRSΔCD) that deleted the entire CD, and joined an N-terminal WHEP to the C-terminal anticodon-binding domain (ABD). X-ray crystallography and three-dimensional NMR revealed the structures of human HisRS and HisRSΔCD. In contrast to homodimeric HisRS, HisRSΔCD is monomeric, where rupture of the ABD's packing with CD resulted in a dumbbell-like structure of flexibly linked WHEP and ABD domains. In addition, the ABD of HisRSΔCD presents a distinct local conformation. This natural internally deleted HisRS suggests evolutionary pressure to reshape AARS tertiary and quaternary structures for repurposing.     

WHEP domains direct noncanonical function of glutamyl-Prolyl tRNA synthetase in translational control of gene expression

The heterotetrameric GAIT complex suppresses translation of selected mRNAs in interferon-gamma-activated monocytic cells. Specificity is dictated by glutamyl-prolyl tRNA synthetase (EPRS) binding to a 3'UTR element in target mRNAs. EPRS consists of two synthetase cores joined by a linker containing three WHEP domains of unknown function. Here we show the critical role of EPRS WHEP domains in targeting and regulating GAIT complex binding to RNA. The upstream WHEP pair directs high-affinity binding to GAIT element-bearing mRNAs, while the overlapping, downstream pair binds NSAP1, which inhibits mRNA binding. Interaction of EPRS with ribosomal protein L13a and GAPDH induces a conformational switch that rescues mRNA binding and restores translational control. Total reconstitution from purified components indicates that the four GAIT proteins are necessary and sufficient for self-assembly of a functional complex. Our results establish the essentiality of WHEP domains in the noncanonical function of EPRS in regulating inflammatory gene expression.     

Cancer-associated splicing variant of tumor suppressor AIMP2/p38: pathological implication in tumorigenesis

Although ARS-interacting multifunctional protein 2 (AIMP2, also named as MSC p38) was first found as a component for a macromolecular tRNA synthetase complex, it was recently discovered to dissociate from the complex and work as a potent tumor suppressor. Upon DNA damage, AIMP2 promotes apoptosis through the protective interaction with p53. However, it was not demonstrated whether AIMP2 was indeed pathologically linked to human cancer. In this work, we found that a splicing variant of AIMP2 lacking exon 2 (AIMP2-DX2) is highly expressed by alternative splicing in human lung cancer cells and patient's tissues. AIMP2-DX2 compromised pro-apoptotic activity of normal AIMP2 through the competitive binding to p53. The cells with higher level of AIMP2-DX2 showed higher propensity to form anchorage-independent colonies and increased resistance to cell death. Mice constitutively expressing this variant showed increased susceptibility to carcinogen-induced lung tumorigenesis. The expression ratio of AIMP2-DX2 to normal AIMP2 was increased according to lung cancer stage and showed a positive correlation with the survival of patients. Thus, this work identified an oncogenic splicing variant of a tumor suppressor, AIMP2/p38, and suggests its potential for anti-cancer target.     

Heat shock protein 90 mediates protein-protein interactions between human aminoacyl-tRNA synthetases

Heat shock protein 90 (hsp90) is a molecular chaperone responsible for protein folding and maturation in vivo. Interaction of hsp90 with human glutamyl-prolyl-tRNA synthetase (EPRS) was found by genetic screening, co-immunoprecipitation, and in vitro binding experiments. This interaction was sensitive to the hsp90 inhibitor, geldanamycin, and also ATP, suggesting that the chaperone activity of hsp90 is required for interaction with EPRS. Interaction of EPRS with hsp90 was targeted to the region of three tandem repeats linking the two catalytic domains of EPRS that is also responsible for the interaction with isoleucyl-tRNA synthetase (IRS). Interaction of EPRS and IRS also depended on the activity of hsp90, implying that their association was mediated by hsp90. EPRS and IRS form a macromolecular protein complex with at least six other tRNA synthetases and three cofactors. hsp90 preferentially binds to most of the complex-forming enzymes rather than those that are not found in the complex. In addition, inactivation of hsp90 interfered with the in vivo incorporation of the nascent aminoacyl-tRNA synthetases into the multi-ARS complex. Thus, hsp90 appears to mediate protein-protein interactions of mammalian tRNA synthetases.     

Substrate Specificity and Oligomerization of Human GMP Synthetase

Guanine monophosphate (GMP) synthetase is a bifunctional two-domain enzyme. The N-terminal glutaminase domain generates ammonia from glutamine and the C-terminal synthetase domain aminates xanthine monophosphate (XMP) to form GMP. Mammalian GMP synthetases (GMPSs) contain a 130-residue-long insert in the synthetase domain in comparison to bacterial proteins. We report here the structure of a eukaryotic GMPS. Substrate XMP was bound in the crystal structure of the human GMPS enzyme. XMP is bound to the synthetase domain and covered by a LID motif. The enzyme forms a dimer in the crystal structure with subunit orientations entirely different from the bacterial counterparts. The inserted sub-domain is shown to be involved in substrate binding and dimerization. Furthermore, the structural basis for XMP recognition is revealed as well as a potential allosteric site. Enzymes in the nucleotide metabolism typically display an increased activity in proliferating cells due to the increased need for nucleotides. Many drugs used as immunosuppressants and for treatment of cancer and viral diseases are indeed nucleobase- and nucleoside-based compounds, which are acting on or are activated by enzymes in this pathway. The information obtained from the crystal structure of human GMPS might therefore aid in understanding interactions of nucleoside-based drugs with GMPS and in structure-based design of GMPS-specific inhibitors.     

Structural separation of different extracellular activities in aminoacyl-tRNA synthetase-interacting multi-functional protein, p43/AIMP1

AIMP1 (previously known as p43) is first found as a factor associated with a macromolecular tRNA synthetase complex. However, it is also secreted and acts on diverse target cells such as endothelial cells, macrophages, and fibroblasts to control angiogenesis, inflammation, and dermal regeneration, respectively. We previously showed that AIMP1 induces the death of endothelial cell but proliferation of fibroblasts and activates macrophages. In this work, we found that elastase 2-cleaved AIMP1 retained its pro-apoptotic activity to endothelial cells but lost the growth-stimulatory activity to fibroblasts. To determine the functional domains responsible for each activity, we generated several deletion fragments of AIMP1 and compared the activities to the target cells. AIMP1 promoted endothelial cell death and caspase-3 activation through its 101-114 amino acid region, fibroblast proliferation through its 6-46 amino acid region, and endothelial migration through its 114-192 amino acid region as revealed by deletion mapping. Thus, this work revealed that AIMP1 uses different regions for its diverse extracellular activities.     

Comparison of the Intrinsic Dynamics of Aminoacyl-tRNA Synthetases

Aminoacyl-tRNA synthetases (AARSs) are an important family of enzymes that catalyze tRNA aminoacylation reaction (Ibba and Soll in Annu Rev Biochem 2000, 69:617–650) [1]. AARSs are grouped into two broad classes (class I and II) based on sequence/structural homology and mode of their interactions with the tRNA molecule (Ibba and Soll in Annu Rev Biochem 2000, 69:617–650) [1]. As protein dynamics play an important role in enzyme function, we explored the intrinsic dynamics of these enzymes using normal mode analysis and investigated if the two classes and six subclasses (Ia–c and IIa–c) of AARSs exhibit any distinct patterns of motion. The present study found that the intrinsic dynamics-based classification of these enzymes is similar to that obtained based on sequence/structural homology for most enzymes. However, the classification of seryl-tRNA synthetase was not straightforward; the internal mobility patterns of this enzyme are comparable to both IIa and IIb AARSs. This study revealed only a few general mobility patterns in these enzymes—(1) the insertion domain is generally engaged in anticorrelated motion with respect to the catalytic domain for both classes of AARSs and (2) anticodon binding domain dynamics are partly correlated and partly anticorrelated with respect to other domains for class I enzymes. In most of the class II AARSs, the anticodon binding domain is predominately engaged in anticorrelated motion with respect to the catalytic domain and correlated to the insertion domain. This study supports the notion that dynamic-based classification could be useful for functional classification of proteins.     

Degradation of AIMP1/p43 Induced by Hepatitis C Virus E2 Leads to Upregulation of TGF-β Signaling and Increase in Surface Expression of gp96

Hepatitis C virus (HCV) causes chronic hepatitis leading to liver fibrosis and autoimmune diseases. AIMP1/p43 is a multifunctional protein initially known as a cofactor of aminoacyl tRNA synthetase complex. Its function includes negative regulation of TGF-β signaling and suppression of Lupus-like autoimmune disease by inhibition of surface expression of gp96. HCV E2 was shown to directly interact with AIMP1/p43 by GST pulldown assay and coimmunoprecipitation. Their subcellular colocalization was observed in an immunofluorescence confocal microscopy. We showed that HCV E2 led to degradation of AIMP1/p43 in two ways. First, in the presence of HCV E2, endogenous AIMP1/p43 was shown to be degraded in an ubiquitin-dependent proteasome pathway. Second, grp78, an ER chaperone, was shown to interact with and stabilize AIMP1/p43. And HCV E2 inhibited this interaction leading to reduction of cellular AIMP1/p43. The degradation of AIMP1/p43 by HCV E2 resulted in increase of TGF-β signaling and cell surface expression of gp96. Thus we suggest that these are novel mechanisms responsible for liver fibrosis and autoimmune diseases caused by HCV.     

Disease-associated mutations in a bifunctional aminoacyl-tRNA synthetase gene elicit the integrated stress response

Aminoacyl-tRNA synthetases (ARSs) catalyze the charging of specific amino acids onto cognate tRNAs, an essential process for protein synthesis. Mutations in ARSs are frequently associated with a variety of human diseases. The human EPRS1 gene encodes a bifunctional glutamyl-prolyl-tRNA synthetase (EPRS) with two catalytic cores and appended domains that contribute to nontranslational functions. In this study, we report compound heterozygous mutations in EPRS1, which lead to amino acid substitutions P14R and E205G in two patients with diabetes and bone diseases. While neither mutation affects tRNA binding or association of EPRS with the multisynthetase complex, E205G in the glutamyl-tRNA synthetase (ERS) region of EPRS is defective in amino acid activation and tRNA^Glu charging. The P14R mutation induces a conformational change and altered tRNA charging kinetics in vitro. We propose that the altered catalytic activity and conformational changes in the EPRS variants sensitize patient cells to stress, triggering an increased integrated stress response (ISR) that diminishes cell viability. Indeed, patient-derived cells expressing the compound heterozygous EPRS show heightened induction of the ISR, suggestive of disruptions in protein homeostasis. These results have important implications for understanding ARS-associated human disease mechanisms and development of new therapeutics.     

Yeast tRNAAsp-Aspartyl-tRNA Synthetase Complex: Low Resolution Crystal Structure

Abstract

Yeast aspartyl-tRNA synthetase, a dimer of molecular weight 125000, and two molecules of its cognate tRNA (M_r = 24160) cocrystallize in the cubic space group 1432 (a = 354 Å). The crystal structure was solved to low resolution using neutron and X-ray diffraction data. Neutron single crystal diffraction data were collected in five solvents differing by their D₂O content in order to use the contrast variation method to distinguish between the protein and tRNA The synthetase was first located at 40 Å resolution using the 65% D₂O neutron data (tRNA matched). tRNA molecules were found at 20 Å resolution using both neutron and X-ray data. The resulting model was refined against 10 Å resolution X-ray data, using density modification and least-squares refinement of the tRNA positions. The crystal structure, solved without a priori phase knowledge, was confirmed later by isomorphous replacement. The molecular model of the complex is in good agreement with results obtained in solution by probing the protected part of the tRNA by chemical reagents.