Mutation analysis of functional role of amino acid residues in domain IV of elongation factor G

Seven variants of elongation factor G (EF-G) from Thermus thermophilus with mutations Glu494Ile, Gly495Asp, Lys496Ile, His509Leu, Lys564Ile and Tyr568Lys located in the beta-sheet of its domain IV and mutation Gly553Asp in a loop between domain III and IV were constructed using polymerase chain reaction. Functional tests demonstrated that only mutation Lys496Ile, located in the vicinity of the loop 501-504, inhibits translocation effectiveness, in the presence of the mutated EF-G. The functional analysis of all mutations constructed up to now in domain IV reveals that only those located in loops 501-504 and 573-578 markedly decrease the translocation activity of EF-G. These loops are located at the tip of domain IV and close to the decoding center of the 30S ribosomal subunit upon EF-G interaction with the ribosome. The functional role of EF-G and its domain IV in ribosomal translocation is discussed.     

Mutation Analysis of the Role in Ribosomal Translocation for Loops of Domain IV of the Elongation Factor G

Seven variants of Thermus thermophilus elongation factor G (EF-G) with mutations in loops of domain IV were constructed by PCR. Point mutations Arg504 Thr, Pro554 Thr, or Ile534 Asp did not affect the GTPase and translocational activities of EF-G. Similar results were obtained for mutants with tetra- or hexapeptide inserts in two loops located at the tip and two loops at the base of domain IV. Insertion of tetrapeptide Gly-Ser-Gly-Thr into loop 501–504 at the tip of domain IV dramatically reduced the activity of EF-G in poly(U)-directed polyphenylalanine synthesis on ribosomes, and halved its translocational activity. The intact conformation of loop Thr501-Gly-Gly-Arg504 was assumed to be essential for sterically perfect, efficient interaction of EF-G with the ribosome. The structural and biochemical data on the 30S subunit and EF-G were analyzed to specify the position of EF-G relative to the 30S and 50S ribosomal subunits.     

The function of conserved amino acid residues adjacent to the effector domain in elongation factor G

Bacterial elongation factor G (EF-G) physically associates with translocation-competent ribosomes and facilitates transition to the subsequent codon through the coordinate binding and hydrolysis of GTP. In order to investigate the amino acid positions necessary for EF-G functions, a series of mutations were constructed in the EF-G structural gene (fusA) of Escherichia coli, specifically at positions flanking the effector domain. A mutated allele was isolated in which the wild-type sequence from codons 29 to 47 ("EFG2947") was replaced with a sequence encoding 28 amino acids from ribosomal protein S7. This mutated gene was unable to complement a fusAts strain when supplied in trans at the nonpermissive temperature. In vitro biochemical analysis demonstrated that nucleotide crosslinking was unaffected in EFG2947, while ribosome binding appeared to be completely abolished. A series of point mutations created within this region, encoding L30A, Y32A, H37A, and K38A were shown to give rise to fully functional proteins, suggesting that side chains of these individual residues are not essential for EF-G function. A sixth mutant, E41A, was found to inefficiently rescue growth in a fusAts background, and was also unable to bind ribosomes normally in vitro. In contrast E41Q could restore growth at the nonpermissive temperature. These results can be explained within the context of a three-dimensional model for the effector region of EF-G. This model indicates that the effector domain contains a negative potential field that may be important for ribosome binding.     

Directed mutagenesis identifies amino acid residues involved in elongation factor Tu binding to yeast Phe-tRNAPhe

The co-crystal structure of Thermus aquaticus elongation factor Tu.guanosine 5'- [beta,gamma-imido]triphosphate (EF-Tu.GDPNP) bound to yeast Phe-tRNA(Phe) reveals that EF-Tu interacts with the tRNA body primarily through contacts with the phosphodiester backbone. Twenty amino acids in the tRNA binding cleft of Thermus Thermophilus EF-Tu were each mutated to structurally conservative alternatives and the affinities of the mutant proteins to yeast Phe-tRNA(Phe) determined. Eleven of the 20 mutations reduced the binding affinity from fourfold to >100-fold, while the remaining ten had no effect. The thermodynamically important residues were spread over the entire tRNA binding interface, but were concentrated in the region which contacts the tRNA T-stem. Most of the data could be reconciled by considering the crystal structures of both free EF-Tu.GTP and the ternary complex and allowing for small (1.0 A) movements in the amino acid side-chains. Thus, despite the non-physiological crystallization conditions and crystal lattice interactions, the crystal structures reflect the biochemically relevant interaction in solution.     

Crystal structure of a mutant elongation factor G trapped with a GTP analogue

Elongation factor G (EF-G) is a G protein factor that catalyzes the translocation step in protein synthesis on the ribosome. Its GTP conformation in the absence of the ribosome is currently unknown. We present the structure of a mutant EF-G (T84A) in complex with the non-hydrolysable GTP analogue GDPNP. The crystal structure provides a first insight into conformational changes induced in EF-G by GTP. Comparison of this structure with that of EF-G in complex with GDP suggests that the GTP and GDP conformations in solution are very similar and that the major contribution to the active GTPase conformation, which is quite different, therefore comes from its interaction with the ribosome.     

Structural analysis of the elongation factor G protein from the low-temperature-adapted bacterium Arthrobacter globiformis SI55

The first structural analysis of elongation factor G (EF-G) from a cold-adapted bacterium is presented. EF-G is an essential protein involved in the elongation process during protein synthesis and is therefore thought to play a crucial role in the low-temperature adaptation of cold-adapted microorganisms. To define its importance, the EF-G gene (fus) from the psychrotolerant bacterium Arthrobacter globiformis SI55 was cloned and sequenced. The deduced primary structure of the elongation factor is composed of 700 amino acids with a predicted molecular mass of 77.4 kDa. A three-dimensional model of the protein was constructed based on the known crystal structures of structurally homologous proteins. Structural features that might potentially be important for activity and flexibility at low temperature were deduced by comparisons with models of the EF-G proteins from the closely related mesophiles Micrococcus luteus and Mycobacterium tuberculosis. These features include a loss in the number of salt bridges in intradomain and interdomain positions, increased solvent interactions mediated by greater charge and polarity on domain surfaces, loop insertions, loss of proline residues in loop structures, and an increase of hydrophobicity in core regions. Specific changes have also been identified in the catalytic domain (G domain) and sites of potential ribosome interaction, which may directly affect guanosine triphosphate (GTP) hydrolysis and elongation rates at low temperature. Received: September 20, 1999 / Accepted: December 2, 1999     

The uncharacterized bacterial protein YejG has the same architecture as domain III of elongation factor G

InterPro family IPR020489 comprises ~1000 uncharacterized bacterial proteins. Previously we showed that overexpressing the Escherichia coli representative of this family, EcYejG, conferred low-level resistance to aminoglycoside antibiotics. In an attempt to shed light on the biochemical function of EcYejG, we have solved its structure using multinuclear solution NMR spectroscopy. The structure most closely resembles that of domain III from elongation factor G (EF-G). EF-G catalyzes ribosomal translocation and mutations in EF-G have also been associated with aminoglycoside resistance. While we were unable to demonstrate a direct interaction between EcYejG and the ribosome, the protein might play a role in translation.     

Kirromycin-resistant elongation factor Tu from pulvomycin-producing wild-type of streptoverticillium mobaraense

Elongation factor Tu (EF-Tu) ofStreptoverticillium mobaraense, which produces pulvomycin, has been prepared to 90% purity. The purified protein differs significantly from the analogous protein found inEscherichia coli in molecular weight and antibiotic sensitivity. EF-Tu migrates in sodium dodecyl sulfate gel electrophoresis as a 46,000-dalton species. The protein is sensitive to pulvomycin, but highly resistant to kirromycin. EF-Tu fromStv. mobaraense exists in multiple forms as monomer and polymers. By contrast to the monomer, the polymers of EF-Tu are completely resistant to pulvomycin.     

Carboxyl-terminal amino acid residues in elongation factor G essential for ribosome association and translocation.

The translocation of ribosomes on mRNA is carried out by cellular machinery that has been extremely well conserved across the entire spectrum of living species. This process requires elongation factor G (EF-G, or EF-2 in archaebacteria and eukaryotes), which is a member of the GTPase superfamily. Using genetic techniques, we have identified a series of mutated alleles of fusA (the Escherichia coli gene that encodes EF-G) that were unable to support protein synthesis in vivo. These alleles encode proteins with point mutations at codons 495 (a variant with a Q-to-P change at codon 495 [Q495P]), 502 (G502D), and 563 (G563D) and a nonsense mutation at codon 608. Biochemical analyses demonstrated that EF-G Q495P, G502D, and delta 608-703 were not disrupted in guanine nucleotide binding but were deficient in ribosome-dependent GTP hydrolysis and guanine nucleotide-dependent ribosome association. We propose that all of these mutations are present in a domain that is essential for ribosome association and that GTP hydrolysis was deficient as a secondary consequence of impaired binding to 70S ribosomes.     

Identification of amino acid residues essential for heparin binding by the A1 domain of human von Willebrand factor

Platelet adhesion is mediated by von Willebrand factor (VWF) that binds platelet glycoprotein Ib (GPIb). Previous observations suggested that heparin competitively inhibits the binding of VWF to GPIb and may down-regulate platelet adhesion. We performed charged-to-alanine scanning mutagenesis of domain A1 and studied dose-dependent binding to heparin-Sepharose beads. Mutations at Lys1362 and Arg1395, at which the GPIb binding was markedly decreased, showed 41% and 42% binding, respectively. Clustered mutations in the segments 1332KDRKR1336 and 1405KKKK1408, which have been proposed as heparin binding sequences, showed 72% and 52% binding, respectively. However, single alanine substitutions within these clusters showed normal binding. Our findings suggest that heparin may inhibit the binding of VWF to GPIb by interacting with GPIb binding and interpret why some hemorrhagic complications of heparin therapy are not predictable based on techniques for monitoring the conventional anticoagulant effects of heparin.     

Initiation factor IF2, thiostrepton and micrococcin prevent the binding of elongation factor G to the Escherichia coli ribosome

The bacterial translational GTPases (initiation factor IF2, elongation factors EF-G and EF-Tu and release factor RF3) are involved in all stages of translation, and evidence indicates that they bind to overlapping sites on the ribosome, whereupon GTP hydrolysis is triggered. We provide evidence for a common ribosomal binding site for EF-G and IF2. IF2 prevents the binding of EF-G to the ribosome, as shown by Western blot analysis and fusidic acid-stabilized EF-G.GDP.ribosome complex formation. Additionally, IF2 inhibits EF-G-dependent GTP hydrolysis on 70 S ribosomes. The antibiotics thiostrepton and micrococcin, which bind to part of the EF-G binding site and interfere with the function of the factor, also affect the function of IF2. While thiostrepton is a strong inhibitor of EF-G-dependent GTP hydrolysis, GTP hydrolysis by IF2 is stimulated by the drug. Micrococcin stimulates GTP hydrolysis by both factors. We show directly that these drugs act by destabilizing the interaction of EF-G with the ribosome, and provide evidence that they have similar effects on IF2.     

Structure and function of FusB: an elongation factor G-binding fusidic acid resistance protein active in ribosomal translocation and recycling

Fusidic acid (FA) is a bacteriostatic antibiotic that locks elongation factor G (EF-G) to the ribosome after GTP hydrolysis during elongation and ribosome recycling. The plasmid pUB101-encoded protein FusB causes FA resistance in clinical isolates of Staphylococcus aureus through an interaction with EF-G. Here, we report 1.6 and 2.3 Å crystal structures of FusB. We show that FusB is a two-domain protein lacking homology to known structures, where the N-terminal domain is a four-helix bundle and the C-terminal domain has an alpha/beta fold containing a C4 treble clef zinc finger motif and two loop regions with conserved basic residues. Using hybrid constructs between S. aureus EF-G that binds to FusB and Escherichia coli EF-G that does not, we show that the sequence determinants for FusB recognition reside in domain IV and involve the C-terminal helix of S. aureus EF-G. Further, using kinetic assays in a reconstituted translation system, we demonstrate that FusB can rescue FA inhibition of tRNA translocation as well as ribosome recycling. We propose that FusB rescues S. aureus from FA inhibition by preventing formation or facilitating dissociation of the FA-locked EF-G–ribosome complex.     

Distinct functions of elongation factor G in ribosome recycling and translocation

Elongation factor G (EF-G) promotes the translocation step in bacterial protein synthesis and, together with ribosome recycling factor (RRF), the disassembly of the post-termination ribosome. Unlike translocation, ribosome disassembly strictly requires GTP hydrolysis by EF-G. Here we report that ribosome disassembly is strongly inhibited by vanadate, an analog of inorganic phosphate (Pi), indicating that Pi release is required for ribosome disassembly. In contrast, the function of EF-G in single-round translocation is not affected by vanadate, while the turnover reaction is strongly inhibited. We also show that the antibiotic fusidic acid blocks ribosome disassembly by EF-G/RRF at a 1000-fold lower concentration than required for the inhibition of EF-G turnover in vitro and close to the effective inhibitory concentration in vivo, suggesting that the antimicrobial activity of fusidic acid is primarily due to the direct inhibition of ribosome recycling. Our results indicate that conformational coupling between EF-G and the ribosome is principally different in translocation and ribosome disassembly. Pi release is not required for the mechanochemical function of EF-G in translocation, whereas the interactions between RRF and EF-G introduce tight coupling between the conformational change of EF-G induced by Pi release and ribosome disassembly.     

Mutational analysis of the functional role of the loop region in the elongation factor G fourth domain in the ribosomal translocation

Seven variants of Thermus thermophilus elongation factor G (EF-G) with mutations in loops of domain IV were constructed by PCR. Point mutations Arg504-->Thr, Pro554-->Thr, or Ile534-->Asp did not affect the GTPase and translocational activities of EF-G. Similar results were obtained for mutants with tetra- or hexapeptide inserts in two loops located at the tip and two loops at the base of domain IV. Insertion of tetrapeptide Gly-Ser-Gly-Thr into loop 501--504 at the tip of domain IV dramatically reduced the activity of EF-G in poly(U)-directed polyphenylalanine synthesis on ribosomes, and halved its translocational activity. The intact conformation of loop Thr501-Gly-Gly-Arg504 was assumed to be essential for sterically perfect, efficient interaction of EF-G with the ribosome. The structural and biochemical data on the 30S subunit and EF-G were analyzed to specify the position of EF-G relative to the 30S and 50S ribosomal subunits.     

Interactions of fusidic acid and elongation factor G with lipid membranes

Fusidic acid (FA) is a potent antibiotic and blocks the protein synthesis by binding to elongation factor G (EF-G) directly. Here we hypothesized that the antibiotic activity of FA would be potentiated by several orders of magnitude if both FA and EF-G would be residing in the lipid membranes and, hence, the probability of interaction would transform from three-dimensional to two-dimensional. Such detailed information could lead to more effective therapeutic interventions if they are understood on a molecular level. Interactions between FA and various lipid membranes composed of 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) and cholesterol (Chol) were studied by capillary electrochromatography (CEC). The influence of the lipid vesicle size--sonicated liposomes and liposomes extruded through 30-, 50-, and 100-nm filters--on the packing of vesicles on the silica capillary surface was investigated by CEC and dissipative quartz crystal microbalance. The CEC results evidenced that FA interacts with and resides in phospholipid membranes. Likewise, monolayer, asymmetrical flow field flow fractionation, and CEC studies confirmed that EF-G is hydrophobic and incorporated into POPC and POPC/Chol membranes. Including EF-G in phospholipid vesicles did not improve the binding of FA to the membranes.     

Enhancement of translation elongation in neurons by brain-derived neurotrophic factor: implications for mammalian target of rapamycin signaling

The effects and signaling mechanisms of brain-derived neurotrophic factor (BDNF) on translation elongation were investigated in cortical neurons. BDNF increased the elongation rate approximately twofold, as determined by measuring the ribosomal transit time. BDNF-accelerated elongation was inhibited by rapamycin, implicating the mammalian target of rapamycin (mTOR). To explore the mechanisms underlying these effects, we examined the protein phosphorylation cascades that lead to the activation of translation elongation in neurons. BDNF increased eukaryote elongation factor 1A (eEF1A) phosphorylation and decreased eEF2 phosphorylation. Whereas eEF2 phosphorylation levels altered by BDNF were inhibited by rapamycin, eEF1A phosphorylation was not affected by rapamycin or PD98059, a mitogen-activated protein kinase kinase (MEK) inhibitor. BDNF induced phosphorylation of eEF2 kinase (Ser366), as well as decreased its kinase activity. All these events were inhibited by rapamycin. Furthermore, mTOR siRNA, which reduced mTOR levels up to 50%, inhibited the BDNF-induced enhancement in elongation rate and decrease in eEF2 phosphorylation. These results strongly suggest that BDNF enhances translation elongation through the activation of the mTOR-eEF2 pathway.     

GTPase activation of elongation factor EF‐Tu by the ribosome during decoding

We have used single‐particle reconstruction in cryo‐electron microscopy to determine a structure of the Thermus thermophilus ribosome in which the ternary complex of elongation factor Tu (EF‐Tu), tRNA and guanine nucleotide has been trapped on the ribosome using the antibiotic kirromycin. This represents the state in the decoding process just after codon recognition by tRNA and the resulting GTP hydrolysis by EF‐Tu, but before the release of EF‐Tu from the ribosome. Progress in sample purification and image processing made it possible to reach a resolution of 6.4 Å. Secondary structure elements in tRNA, EF‐Tu and the ribosome, and even GDP and kirromycin, could all be visualized directly. The structure reveals a complex conformational rearrangement of the tRNA in the A/T state and the interactions with the functionally important switch regions of EF‐Tu crucial to GTP hydrolysis. Thus, the structure provides insights into the molecular mechanism of signalling codon recognition from the decoding centre of the 30S subunit to the GTPase centre of EF‐Tu.     

Immunocytochemical localization of the elongation factor Tu in E. coli cells

The localization of the elongation factor Tu (EF-Tu) in ultrathin cryosections of E. coli cells was determined with the electron microscope using a highly specific immunological labelling technique. EF-Tu is distributed almost homogeneously throughout the cytoplasm. Although it has often been suggested that EF-Tu could be part of a putative prokaryotic cytoskeleton, we did not find any evidence for supramolecular assemblies, such as fibres or filaments, containing a large amount of EF-Tu. EF-Tu was not observed in association with the outer cell membrane and periplasmic space. A topological relationship with the inner membrane is not apparent in our micrographs. In cells in which the EF-Tu level is raised significantly, the protein piles up in discrete cell regions.