Elongation factor 1A1 regulates metabolic substrate preference in mammalian cells.

Eukaryotic elongation factor 1A1 (EEF1A1) is canonically involved in protein synthesis but also has noncanonical functions in diverse cellular processes. Previously, we identified EEF1A1 as a mediator of lipotoxicity and demonstrated that chemical inhibition of EEF1A1 activity reduced mouse liver lipid accumulation. These findings suggested a link between EEF1A1 and metabolism. Therefore, we investigated its role in regulating metabolic substrate preference. EEF1A1-deficient Chinese hamster ovary (2E2) cells displayed reduced media lactate accumulation. These effects were also observed with EEF1A1 knockdown in human hepatocyte-like HepG2 cells and in WT Chinese hamster ovary and HepG2 cells treated with selective EEF1A inhibitors, didemnin B, or plitidepsin. Extracellular flux analyses revealed decreased glycolytic ATP production and increased mitochondrial-to-glycolytic ATP production ratio in 2E2 cells, suggesting a more oxidative metabolic phenotype. Correspondingly, fatty acid oxidation was increased in 2E2 cells. Both 2E2 cells and HepG2 cells treated with didemnin B exhibited increased neutral lipid content, which may be required to support elevated oxidative metabolism. RNA-seq revealed a >90-fold downregulation of a rate-limiting glycolytic enzyme, hexokinase 2, which we confirmed through immunoblotting and enzyme activity assays. Pathway enrichment analysis identified downregulations in TNFA signaling via NFKB and MYC targets. Correspondingly, nuclear abundances of RELB and MYC were reduced in 2E2 cells. Thus, EEF1A1 deficiency may perturb glycolysis by limiting NFKB- and MYC-mediated gene expression, leading to decreased hexokinase expression and activity. This is the first evidence of a role for a translation elongation factor, EEF1A1, in regulating metabolic substrate utilization in mammalian cells.

[Role of Eukaryotic Translation Elongation Factor 1 Family Members in the Tumorigenesis and Progression of Lung Adenocarcinoma].

Objective To investigate the role and mechanism of eukaryotic translation elongation factor 1(EEF1) family members (EEF1D,EEF1A1,and EEF1A2) in lung adenocarcinoma (LUAD) based on public databases.Methods We examined EEF1 member expression levels in human LUAD samples via The Cancer Genome Atlas in the UCSC Xena browser and the Clinical Proteomic Tumor Analysis Consortium.We analyzed the mRNA and protein levels of EEF1D,EEF1A1,and EEF1A2 and their correlations with pathological variables via the Mann-Whitney U test.The Kaplan-Meier curves were established to assess the prognostic values of EEF1D,EEF1A1,and EEF1A2.The single-sample gene set enrichment analysis algorithm was employed to explore the relationship between the expression levels of EEF1 members and tumor immune cell infiltration.Spearman and Pearson correlation analyses were performed to examine the relationship between the expression levels of EEF1 members and those of the genes in the phosphatidylinositol 3-kinase/protein kinase B signaling pathway.The immunohistochemical assay was employed to determine the expression levels of EEF1D,EEF1A1,and EEF1A2 in the LUAD tissue (n=75) and paracancer tissue (n=75) samples.Results The mRNA and protein levels of EEF1D,EEF1A1,and EEF1A2 showed significant differences between tumor and paracancer tissues (all P<0.001).The patients with high protein levels of EEF1A1 showed bad prognosis in terms of overall survival (P=0.039),and those with high protein levels of EEF1A2 showed good prognosis in terms of overall survival (P=0.012).The influence of the mRNA level of EEF1D on prognosis was associated with pathological characteristics.The expression levels of EEF1 members were significantly associated with the infiltration of various immune cells and the expression of key molecules in the phosphatidylinositol 3-kinase/protein kinase B signaling pathway.Conclusion EEF1D,EEF1A1,and EEF1A2 are associated with the progression of LUAD,serving as the candidate prognostic markers for LUAD.

Probe Synthesis Reveals Eukaryotic Translation Elongation Factor†1 Alpha†1 as the Anti-Pancreatic Cancer Target of BE-43547A2.

The natural product, BE-43547A2 , decreases pancreatic cancer cell stemness. However, its anticancer molecular mechanisms have not been fully established. Based on structure-activity relationships of BE-43547A2 , we synthesized a probe and investigated its potential targets using an in situ click reaction. We found that BE-43547A2 exerts its anticancer effects by covalently binding the cysteine234 (C234) residue of eukaryotic translation elongation factor 1 alpha 1 (eEF1A1). This binding mode was confirmed by a series of experiments including a xenograft mouse model. We also determined that eEF1A1 plays an important role in regulating pancreatic cancer cell stemness. Analyses of 99 clinical pancreatic cancer samples revealed that eEF1A1 expressions are closely correlated with clinicopathological grade and patient survival. In conclusion, eEF1A1 is involved in pancreatic cancer progression and is therefore, a promising novel covalent target for pancreatic cancer treatment.

Structural Cues for Understanding eEF1A2 Moonlighting.

Spontaneous mutations in the EEF1A2 gene cause epilepsy and severe neurological disabilities in children. The crystal structure of eEF1A2 protein purified from rabbit skeletal muscle reveals a post-translationally modified dimer that provides information about the sites of interaction with numerous binding partners, including itself, and maps these mutations onto the dimer and tetramer interfaces. The spatial locations of the side chain carboxylates of Glu301 and Glu374, to which phosphatidylethanolamine is uniquely attached via an amide bond, define the anchoring points of eEF1A2 to cellular membranes and interorganellar membrane contact sites. Additional bioinformatic and molecular modeling results provide novel structural insight into the demonstrated binding of eEF1A2 to SH3 domains, the common MAPK docking groove, filamentous actin, and phosphatidylinositol-4 kinase IIIß. In this new light, the role of eEF1A2 as an ancient, multifaceted, and articulated G protein at the crossroads of autophagy, oncogenesis and viral replication appears very distant from the "canonical" one of delivering aminoacyl-tRNAs to the ribosome that has dominated the scene and much of the thinking for many decades.

Expression of Hybrid Peptide EF-1 in Pichia pastoris, Its Purification, and Antimicrobial Characterization.

EF-1 is a novel peptide derived from two bacteriocins, plantaricin E and plantaricin F. It has a strong antibacterial activity against Escherichia coli and with negligible hemolytic effect on red blood cells. However, the chemical synthesis of EF-1 is limited by its high cost. In this study, we established a heterologous expression of EF-1 in Pichia pastoris. The transgenic strain successfully expressed hybrid EF-1 peptide, which had a molecular weight of ~5 kDa as expected. The recombinant EF-1 was purified by Ni2+ affinity chromatography and reversed-phase high performance liquid chromatography (RP-HPLC), which achieved a yield of 32.65 mg/L with a purity of 94.9%. The purified EF-1 exhibited strong antimicrobial and bactericidal activities against both Gram-positive and -negative bacteria. Furthermore, propidium iodide staining and scanning electron microscopy revealed that EF-1 can directly induce cell membrane permeabilization of E. coli. Therefore, the hybrid EF-1 not only preserves the individual properties of the parent peptides, but also acquires the ability to disrupt Gram-negative bacterial membrane. Meanwhile, such an expression system can reduce both the time and cost for large-scale peptide production, which ensures its potential application at the industrial level.

The protein-binding N-terminal domain of human translation elongation factor 1Bß possesses a dynamic α-helical structural organization.

Translation elongation factor 1Bß (eEF1Bß) is a metazoan-specific protein involved into the macromolecular eEF1B complex, containing also eEF1Bα and eEF1Bγ subunits. Both eEF1Bα and eEF1Bß ensure the guanine nucleotide exchange on eEF1A while eEF1Bγ is thought to have a structural role. The structures of the eEF1Bß catalytic C-terminal domain and neighboring central acidic region are known while the structure of the protein-binding N-terminal domain remains unidentified which prevents clear understanding of architecture of the eEF1B complex. Here we show that the N-terminal domain comprising initial 77 amino acids of eEF1Bß, eEF1Bß(1-77), is a monomer in solution with increased hydrodynamic volume. This domain binds eEF1Bγ in equimolar ratio. The CD spectra reveal that the secondary structure of eEF1Bß(1-77) consists predominantly of α-helices and a portion of disordered region. Very rapid hydrogen/deuterium exchange for all eEF1Bß(1-77) peptides favors a flexible tertiary organization of eEF1Bß(1-77). Computational modeling of eEF1Bß(1-77) suggests several conformation states each composed of three α-helices connected by flexible linkers. Altogether, the data imply that the protein-binding domain of eEF1Bß shows flexible spatial organization which may be needed for interaction with eEF1Bγ or other protein partners.

Identification of a Specific Translational Machinery via TCTP-EF1A2 Interaction Regulating NF1-associated Tumor Growth by Affinity Purification and Data-independent Mass Spectrometry Acquisition (AP-DIA).

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease that predisposes individuals to developing benign neurofibromas and malignant peripheral nerve sheath tumors (MPNST). The mechanism of NF1-tumorigenesis or the curatives have not been established. Using unique trascriptome and proteome integration method, iPEACH (1), we previously identified translationally controlled tumor protein (TCTP) as a novel biological target for NF1-associated tumors (2). Here, we identified specific TCTP-interacting proteins by sequential affinity purification and data-independent mass spectrometry acquisition (AP-DIA/SWATH) to investigate the role of TCTP in NF1-associated malignant tumors. TCTP mainly interacts with proteins related to protein synthesis and especially to elongation factor complex components, including EF1A2, EF1B, EF1D, EF1G, and valyl-tRNA synthetase (VARS), in NF1-deficient malignant tumor cells. Interestingly, TCTP preferentially binds to EF1A2 (normally found only in neural and skeletal-muscle cells and several cancer cells), rather than EF1A1 despite the high homologies (98%) in their sequences. The docking simulation and further validations to study the interaction between TCTP and EF1A2 revealed that TCTP directly binds with EF1A2 via the contact areas of EF1A2 dimerization. Using unique and common sequences between EF1A2 and EF1A1 in AP-DIA/SWATH, we quantitatively validated the interaction of EF1A2 and TCTP/other elongation factors and found that TCTP coordinates the translational machinery of elongation factors via the association with EF1A2. These data suggest that TCTP activates EF1A2-dependent translation by mediating complex formation with other elongation factors. Inhibiting the TCTP-EF1A2 interaction with EF1A2 siRNAs or a TCTP inhibitor, artesunate, significantly down-regulated the factors related to protein translation and caused dramatic suppression of growth/translation in NF1-associated tumors. Our findings demonstrate that a specific protein translation machinery related to the TCTP-EF1A2 interaction is functionally implicated in the tumorigenesis and progression of NF1-associated tumors and could represent a therapeutic target.

Airborne transmission of invasive fusariosis in patients with hematologic malignancies.

From 2006 to 2013, an increasing incidence of fusariosis was observed in the hematologic patients of our University Hospital. We suspected of an environmental source, and the indoor hospital air was investigated as a potential source of the fungemia. Air samplings were performed in the hematology and bone marrow transplant (BMT) wards using an air sampler with pre-defined air volumes. To study the molecular relationship among environmental and clinical isolates, 18 Fusarium spp. recovered from blood cultures were included in the study. DNA sequencing of a partial portion of TEF1α gene was performed for molecular identification. Molecular typing was carried out by multi-locus sequence typing (MLST) using a four-gene scheme: TEF1α, rDNA, RPB1 and RPB2. One hundred four isolates were recovered from the air of the hematology (n = 76) and the BMT (n = 28) wards. Fusarium isolates from the air were from five species complexes: Fusarium fujikuroi (FFSC, n = 56), Fusarium incarnatum-equiseti (FIESC, n = 24), Fusarium solani (FSSC, n = 13), Fusarium chlamydosporum (FCSC, n = 10), and Fusarium oxysporum (FOSC, n = 1). Fifteen Fusarium isolates recovered from blood belonged to FSSC, and three to FFSC. MLST identified the same sequence type (ST) in clinical and environmental isolates. ST1 was found in 5 isolates from blood and in 7 from the air, both identified as FSSC (Fusarium petroliphilum). STn1 was found in one isolate from blood and in one from the air, both identified as FFSC (Fusarium napiforme). F. napiforme was isolated from the air of the hospital room of the patient with fungemia due to F. napiforme. These findings suggested a possible clonal origin of the Fusarium spp. recovered from air and bloodcultures. In conclusion, our study found a diversity of Fusarium species in the air of our hospital, and a possible role of the air as source of systemic fusariosis in our immunocompromised patients.

An update on the biophysical character of the human eukaryotic elongation factor 1 beta: Perspectives from interaction with elongation factor 1 gamma.

The ß-subunit of the human eukaryotic elongation factor 1 complex (heEF1ß) plays a central role in the elongation step in eukaryotic protein biosynthesis, which essentially involves interaction with the α- and γ-subunits (eEF1γ). To biophysically characterize heEF1ß, we constructed 3 Escherichia coli expression vector systems for recombinant expression of the full length (FL-heEF1ß), N-terminus (NT-heEF1ß), and the C-terminus (CT-heEF1ß) regions of the protein. Our results suggest that heEF1ß is predominantly alpha-helical and possesses an accessible hydrophobic cavity in the CT-heEF1ß. Both FL-heEF1ß and NT-heEF1ß form dimers of size 62 and 30 kDa, respectively, but the CT-heEF1ß is monomeric. FL-heEF1ß interacts with the N-terminus glutathione transferase-like domain of heEF1γ (NT-heEF1γ) to form a 195-kDa complex or a 230-kDa complex in the presence of oxidized glutathione. On the other hand, NT-heEF1ß forms a 170-kDa complex with NT-heEF1γ and a high molecular weight aggregate of size greater than 670 kDa. Surface plasmon resonance analysis confirmed that (by fitting the Langmuir 1:1 model) FL-heEF1ß associated with monomeric or dimeric NT-heEF1γ at a rapid rate and slowly dissociated, suggesting strong functional affinity (KD  = 9.6 nM for monomeric or 11.3 nM for dimeric NT-heEF1γ). We postulate that the N-terminus region of heEF1ß may be responsible for its dimerization and the C-terminus region of heEF1ß modulates the formation of an ordered heEF1ß-γ oligomer, a structure that may be essential in the elongation step of eukaryotic protein biosynthesis.

Structural rationale for the cross-resistance of tumor cells bearing the A399V variant of elongation factor eEF1A1 to the structurally unrelated didemnin B, ternatin, nannocystin A and ansatrienin B.

At least four classes of structurally distinct natural products with potent antiproliferative activities target the translation elongation factor eEF1A1, which is best known as the G-protein that delivers amino acyl transfer RNAs (aa-tRNAs) to ribosomes during mRNA translation. We present molecular models in atomic detail that provide a common structural basis for the high-affinity binding of didemnin B, ternatin, ansatrienin B and nannocystin A to eEF1A1, as well as a rationale based on molecular dynamics results that accounts for the deleterious effect of replacing alanine 399 with valine. The proposed binding site, at the interface between domains I and III, is eminently hydrophobic and exists only in the GTP-bound conformation. Drug binding at this site is expected to disrupt neither loading of aa-tRNAs nor GTP hydrolysis but would give rise to stabilization of this particular conformational state, in consonance with reported experimental findings. The experimental solution of the three-dimensional structure of mammalian eEF1A1 has proved elusive so far and the highly homologous eEF1A2 from rabbit muscle has been crystallized and solved only as a homodimer in a GDP-bound conformation. Interestingly, in this dimeric structure the large interdomain cavity where the drugs studied are proposed to bind is occupied by a mostly hydrophobic α-helix from domain I of the same monomer. Since binding of this α-helix and any of these drugs to domain III of eEF1A(1/2) is, therefore, mutually exclusive and involves two distinct protein conformations, one intriguing possibility that emerges from our study is that the potent antiproliferative effect of these natural products may arise not only from inhibition of protein synthesis, which is the current dogma, but also from interference with some other non-canonical functions. From this standpoint, this type of drugs could be considered antagonists of eEF1A1/2 oligomerization, a hypothesis that opens up novel areas of research.

Misfolded polypeptides are selectively recognized and transported toward aggresomes by a CED complex.

Misfolded polypeptides are rapidly cleared from cells via the ubiquitin-proteasome system (UPS). However, when the UPS is impaired, misfolded polypeptides form small cytoplasmic aggregates, which are sequestered into an aggresome and ultimately degraded by aggrephagy. Despite the relevance of the aggresome to neurodegenerative proteinopathies, the molecular mechanisms underlying aggresome formation remain unclear. Here we show that the CTIF-eEF1A1-DCTN1 (CED) complex functions in the surveillance of either pre-existing or newly synthesized polypeptides by linking two molecular events: selective recognition and aggresomal targeting of misfolded polypeptides. These events are accompanied by CTIF sequestration into the aggresome, preventing the additional synthesis of misfolded polypeptides from mRNAs bound by nuclear cap-binding complex. These events render cells more resistant to apoptosis induced by proteotoxic stresses. Collectively, our data provide compelling evidence for a previously unappreciated protein surveillance pathway and a regulatory gene expression network for coping with misfolded polypeptides.

Comparison of the ability of mammalian eEF1A1 and its oncogenic variant eEF1A2 to interact with actin and calmodulin.

The question as to why a protein exerts oncogenic properties is answered mainly by well-established ideas that these proteins interfere with cellular signaling pathways. However, the knowledge about structural and functional peculiarities of the oncoproteins causing these effects is far from comprehensive. The 97.5% homologous tissue-specific A1 and A2 isoforms of mammalian translation elongation factor eEF1A represent an interesting model to study a difference between protein variants of a family that differ in oncogenic potential. We propose that the different oncogenic impact of A1 and A2 might be explained by differences in their ability to communicate with their respective cellular partners. Here we probed this hypothesis by studying the interaction of eEF1A with two known partners - calmodulin and actin. Indeed, an inability of the A2 isoform to interact with calmodulin is shown, while calmodulin is capable of binding A1 and interferes with its tRNA-binding and actin-bundling activities in vitro. Both A1 and A2 variants revealed actin-bundling activity; however, the form of bundles formed in the presence of A1 or A2 was distinctly different. Thus, a potential inability of A2 to be controlled by Ca2+-mediated regulatory systems is revealed.

Energetics of Glutathione Binding to Human Eukaryotic Elongation Factor 1 Gamma: Isothermal Titration Calorimetry and Molecular Dynamics Studies.

The energetics of ligand binding to human eukaryotic elongation factor 1 gamma (heEF1γ) was investigated using reduced glutathione (GSH), oxidised glutathione (GSSG), glutathione sulfonate and S-hexylglutathione as ligands. The experiments were conducted using isothermal titration calorimetry, and the findings were supported using computational studies. The data show that the binding of these ligands to heEF1γ is enthalpically favourable and entropically driven (except for the binding of GSSG). The full length heEF1γ binds GSSG with lower affinity (K d = 115 µM), with more hydrogen-bond contacts (ΔH = -73.8 kJ/mol) and unfavourable entropy (-TΔS = 51.7 kJ/mol) compared to the glutathione transferase-like N-terminus domain of heEF1γ, which did not show preference to any specific ligand. Computational free binding energy calculations from the 10 ligand poses show that GSSG and GSH consistently bind heEF1γ, and that both ligands bind at the same site with a folded bioactive conformation. This study reveals the possibility that heEF1γ is a glutathione-binding protein.

Translation elongation factor eEF1A1 is a novel partner of a multifunctional protein Sgt1.

Mammalian translation elongation factor eEF1A is involved in ribosomal polypeptide synthesis. Also, the protein fulfills many additional duties in an eukaryotic cell. Here, we identified a novel partner of the eEF1A1 isoform, namely Sgt1, a protein that possesses co-chaperon properties and participates in antiviral defense processes. By applying different methods, we demonstrated the interaction between eEF1A1 and Sgt1 using both purified proteins and cell lysates. We also found that the D2 and D3 domains of eEF1A1 and the TPR domain of Sgt1 are involved in complex formation. Modeling of the Sgt1-eEF1A1 complex suggested both shape and charge complementarities of the eEF1A1-Sgt1 interface stabilized by a number of salt bridges. As long as such interaction mode is typical more for protein-nucleic acid interaction we suggested a possibility that Sgt1 competes with viral RNA for binding to eEF1A and obtained in vitro evidence to this effect.

Human eukaryotic elongation factor 1A forms oligomers through specific cysteine residues.

Eukaryotic elongation factor 1A (eEF1A) is a multifunctional protein involved in bundling actin, severing microtubule, activating the phosphoinositol-4 kinase, and recruiting aminoacyl-tRNAs to ribosomes during protein biosynthesis. Although evidence has shown the presence of the isoform eEF1A1 oligomers, the substantial mechanism of the self-association remains unclear. Herein, we found that human eEF1A1 could spontaneously form oligomers. Specifically, mutagenesis screen on cysteine residues demonstrated that Cys(234) was essential for eEF1A1 oligomerization. In addition, we also found that hydrogen peroxide treatment could induce the formation of eEF1A oligomers in cells. By cysteine replacement, eEF1A2 isoform displayed the ability to oligomerize in cells under the oxidative environment. In summary, in this study we characterized eEF1A1 oligomerization and demonstrated that specific cysteine residues are required for this oligomerization activity.

Truncation of the A,A(∗),A' helices segment impairs the actin bundling activity of mammalian eEF1A1.

Translation elongation factor eEF1A is a G-protein which has a crucial role in the ribosomal polypeptide elongation and possesses a number of non-translational functions. Here, we show that the A,A(∗),A' helices segment of mammalian eEF1A is dispensable for the eEF1A*eEF1Bα complex formation. The A,A(∗),A' helices region did not interact with actin; however, its removal eliminates the actin bundling activity of eEF1A, probably due to the destruction of a dimeric structure of eEF1A. The translation function of monomers and the actin-bundling function of dimers of mammalian eEF1A is suggested.

Evolutionarily conserved binding of translationally controlled tumor protein to eukaryotic elongation factor 1B.

Translationally controlled tumor protein (TCTP) is an abundant protein that is highly conserved in eukaryotes. However, its primary function is still not clear. Human TCTP interacts with the metazoan-specific eukaryotic elongation factor 1Bδ (eEF1Bδ) and inhibits its guanine nucleotide exchange factor (GEF) activity, but the structural mechanism remains unknown. The interaction between TCTP and eEF1Bδ was investigated by NMR titration, structure determination, paramagnetic relaxation enhancement, site-directed mutagenesis, isothermal titration calorimetry, and HADDOCK docking. We first demonstrated that the catalytic GEF domain of eEF1Bδ is not responsible for binding to TCTP but rather a previously unnoticed central acidic region (CAR) domain in eEF1Bδ. The mutagenesis data and the structural model of the TCTP-eEF1Bδ CAR domain complex revealed the key binding residues. These residues are highly conserved in eukaryotic TCTPs and in eEF1B GEFs, including the eukaryotically conserved eEF1Bα, implying the interaction may be conserved in all eukaryotes. Interactions were confirmed between TCTP and the eEF1Bα CAR domain for human, fission yeast, and unicellular photosynthetic microalgal proteins, suggesting that involvement in protein translation through the conserved interaction with eEF1B represents a primary function of TCTP.

A new type of protein lysine methyltransferase trimethylates Lys-79 of elongation factor 1A.

The elongation factors of Saccharomyces cerevisiae are extensively methylated, containing a total of ten methyllysine residues. Elongation factor methyltransferases (Efm1, Efm2, Efm3, and Efm4) catalyze at least four of these modifications. Here we report the identification of a new type of protein lysine methyltransferase, Efm5 (Ygr001c), which was initially classified as N6-adenine DNA methyltransferase-like. Efm5 is required for trimethylation of Lys-79 on EF1A. We directly show the loss of this modification in efm5Δ strains by both mass spectrometry and amino acid analysis. Close homologs of Efm5 are found in vertebrates, invertebrates, and plants, although some fungal species apparently lack this enzyme. This suggests possible unique functions of this modification in S. cerevisiae and higher eukaryotes. The misannotation of Efm5 was due to the presence of a DPPF sequence in post-Motif II, typically associated with DNA methylation. Further analysis of this motif and others like it demonstrates a potential consensus sequence for N-methyltransferases.

Mammalian translation elongation factor eEF1A2: X-ray structure and new features of GDP/GTP exchange mechanism in higher eukaryotes.

Eukaryotic elongation factor eEF1A transits between the GTP- and GDP-bound conformations during the ribosomal polypeptide chain elongation. eEF1A*GTP establishes a complex with the aminoacyl-tRNA in the A site of the 80S ribosome. Correct codon-anticodon recognition triggers GTP hydrolysis, with subsequent dissociation of eEF1A*GDP from the ribosome. The structures of both the 'GTP'- and 'GDP'-bound conformations of eEF1A are unknown. Thus, the eEF1A-related ribosomal mechanisms were anticipated only by analogy with the bacterial homolog EF-Tu. Here, we report the first crystal structure of the mammalian eEF1A2*GDP complex which indicates major differences in the organization of the nucleotide-binding domain and intramolecular movements of eEF1A compared to EF-Tu. Our results explain the nucleotide exchange mechanism in the mammalian eEF1A and suggest that the first step of eEF1A*GDP dissociation from the 80S ribosome is the rotation of the nucleotide-binding domain observed after GTP hydrolysis.

Different oligomeric properties and stability of highly homologous A1 and proto-oncogenic A2 variants of mammalian translation elongation factor eEF1.

Translation elongation factor 1A (eEF1A) directs aminoacyl-tRNA to the A site of 80S ribosomes. In addition, more than 97% homologous variants of eEF1A, A1 and A2, whose expression in different tissues is mutually exclusive, may fulfill a number of independent moonlighting functions in the cell; for instance, the unusual appearance of A2 in an A1-expressing tissue was recently linked to the induction of carcinogenesis. The structural background explaining the different functional performance of the highly homologous proteins is unclear. Here, the main difference in the structural properties of these proteins was revealed to be the improved ability of A1 to self-associate, as demonstrated by synchrotron small-angle X-ray scattering (SAXS) and analytical ultracentrifugation. Besides, the SAXS measurements at different urea concentrations revealed the low resistance of the A1 protein to urea. Titration of the proteins by hydrophobic dye 8-anilino-1-naphthalenesulfonate showed that the A1 isoform is more hydrophobic than A2. As the different association properties, lipophilicity, and stability of the highly similar eEF1A variants did not influence considerably their translation functions, at least in vitro, we suggest this difference may indicate a structural background for isoform-specific moonlighting roles.