Electron-paramagnetic-resonance studies of manganese(II) complexes with elongation factor Tu from Bacillus stearothermophilus. Observation of a GTP hydrolysis intermediate state complex

Changes in the coordination of Mn2+ to nucleotide, water and protein at the active site of elongation factor Tu (EF-Tu) have been studied by electron paramagnetic resonance (EPR) spectroscopy. From the time dependence of the Mn2+ spectrum after addition of GTP to EF-Tu X Mn, it was apparent that three complexes with different EPR linewidths could be detected. Using additional information from the kinetics of 32Pi production and release from EF-Tu X Mn X [gamma-32P]GTP these were identified as EF-Tu X Mn X GTP (linewidth 4.2 mT), EF-Tu X Mn X GDP X Pi (1.20 mT) and EF-Tu X Mn X GDP (1.29 mT). The linewidth for EF-Tu X Mn was 1.51 mT. The rate constant for GTP cleavage on EF-Tu was 0.01 min-1 at 24 C, for Pi release from the EF-Tu X GDP X Pi complex 0.0033 min-1. The corresponding rate constants in the presence of Mg2+ were 0.003 min-1 and 0.0065 min-1. The rate constant for reversal of the cleavage step was found to be much smaller than that for the rate of Pi release (and consequently much smaller than in the forward direction), as shown by 31P-NMR experiments on the incorporation of 18O into Pi from GTP hydrolyzed in the presence of H2 18O. EPR experiments using specifically 17O-labelled GTPs demonstrated an interaction of Mn2+ with the beta-phosphate in both the EF-Tu X GDP X Pi and EF-Tu X GDP complexes. Inorganic phosphate in the EF-Tu X GDP X Pi complex was found not to interact with the metal ion. From EPR experiments in H2 17O, it was concluded that the most probable number of water molecules in the different complexes was 4 (EF-Tu X Mn), 5 (EF-Tu X Mn X GDP X Pi) and 3 (EF-Tu X Mn X GDP), with 2, 0 and 2 metal-protein interactions respectively.     

Delayed release of inorganic phosphate from elongation factor Tu following GTP hydrolysis on the ribosome

The dissociation of inorganic phosphate (P(i)) following GTP hydrolysis is a key step determining the functional state of many GTPases. Here, the timing of P(i) release from elongation factor Tu (EF-Tu) and its implications for the function of EF-Tu on the ribosome were studied by rapid kinetic techniques. It was found that P(i) release from EF-Tu is >20-fold slower than GTP cleavage and limits the rate of the conformational switch of EF-Tu from the GTP- to the GDP-bound form. The point mutation Gly94Ala in the switch 2 region of EF-Tu abolished the delay in P(i) release, suggesting that P(i) release is controlled by the mobility of the switch 2 region with Gly94 acting as a pivot. The rate of P(i) release or the conformational switch of EF-Tu does not affect the selection of aminoacyl-tRNA on the ribosome. Rather, the slow P(i) release may be a consequence of the tight interaction of the switch regions of EF-Tu with the gamma-phosphate and the ribosome in the GTPase activated state of the factor.     

Nucleotide binding and GTP hydrolysis by elongation factor Tu from Thermus thermophilus as monitored by proton NMR.

Proton NMR experiments of the GTP/GDP-binding protein EF-Tu from the extremely thermophilic bacterium Thermus thermophilus HB8 in H2O have been performed paying special attention to the resonances in the downfield region (below 10 ppm). Most of these downfield signals are due to hydrogen bonds formed between the protein and the bound nucleotide. However, three downfield resonances appear even in the nucleotide-free EF-Tu. The middle and C-terminal domain (domain II/III) of EF-Tu lacking the GTP/GDP-binding domain gives rise to an NMR spectrum that hints at a well-structured protein. In contrast to native EF-Tu, the domain II/III spectrum contains no resonances in the downfield region. Several downfield resonances can be used as a fingerprint to trace hydrolysis of protein-bound GTP and temperature effects on the EF-Tu.GDP spectra. NMR studies of the binding of guanosine nucleotide analogues (GMPPNP, GMPPCP) to nucleotide-free EF-Tu have been carried out. The downfield resonances of these complexes differ from the spectrum of EF-Tu.GTP. Protected and photolabile caged GTP was bound to EF-Tu, and NMR spectra before and after photolysis were recorded. The progress of the GTP hydrolysis could be monitored using this method. The downfield resonances have been tentatively assigned taking into account the known structural and biochemical aspects of EF-Tu nucleotide-binding site.     

Codon-dependent conformational change of elongation factor Tu preceding GTP hydrolysis on the ribosome.

The mechanisms by which elongation factor Tu (EF-Tu) promotes the binding of aminoacyl-tRNA to the A site of the ribosome and, in particular, how GTP hydrolysis by EF-Tu is triggered on the ribosome, are not understood. We report steady-state and time-resolved fluorescence measurements, performed in the Escherichia coli system, in which the interaction of the complex EF-Tu.GTP.Phe-tRNAPhe with the ribosomal A site is monitored by the fluorescence changes of either mant-dGTP [3'-O-(N-methylanthraniloyl)-2-deoxyguanosine triphosphate], replacing GTP in the complex, or of wybutine in the anticodon loop of the tRNA. Additionally, GTP hydrolysis is measured by the quench-flow technique. We find that codon-anticodon interaction induces a rapid rearrangement within the G domain of EF-Tu around the bound nucleotide, which is followed by GTP hydrolysis at an approximately 1.5-fold lower rate. In the presence of kirromycin, the activated conformation of EF-Tu appears to be frozen. The steps following GTP hydrolysis--the switch of EF-Tu to the GDP-bound conformation, the release of aminoacyl-tRNA from EF-Tu to the A site, and the dissociation of EF-Tu-GDP from the ribosome--which are altogether suppressed by kirromycin, are not distinguished kinetically. The results suggest that codon recognition by the ternary complex on the ribosome initiates a series of structural rearrangements resulting in a conformational change of EF-Tu, possibly involving the effector region, which, in turn, triggers GTP hydrolysis.     

The binding of nucleotides and metal ions to elongation factor Tu from Bacillus stearothermophilus as studied by equilibrium dialysis

EF-Tu from B. stearothermophilus binds divalent metal ions even in the absence of guanine nucleotides. The association constants necessary for characterizing the multiple equilibria between EF-Tu, GDP and the divalent ions magnesium and manganese were determined by equilibrium dialysis. The constants are 4.6 X 10(4) M-1 and 5.4 X 10(5) M-1 for the binding of Mg2 and 1.0 X 10(5) M-1 and 1.1 X 10(6) M-1 for the binding of Mn2 to EF-Tu and EF-Tu . GDP, respectively. In the absence of divalent ions EF-Tu binds GMP, GDP and GTP with association constants of 3 x 10(3) M-1, 1.7 x 10(7) M-1 and 1.3 x 10(6) M-1, respectively. The binding of GDP in the presence of metal ions is an order of magnitude stronger than in the absence of metal ions.     

Study of the interaction between uncharged yeast tRNAPhe and elongation factor Tu from Bacillus stearothermophilis

Proton NMR studies are presented on the interaction of nonaminoacylated yeast tRNAPhe and elongation factor Tu X GTP from Bacillus stearothermophilis. From experiments in which transfer of magnetization is observed between proton spins of tRNA and the protein, it is concluded that complex formation takes place. Amino acid residues of the protein come into close contact with the base pair A5U68 and/or U52A62 of the acceptor T psi C limb of the tRNA molecule. From the line broadening of tRNA resonances, associated with complex formation, an association constant of 10(3)-10(4) M-1 is estimated. The NMR experiments do not monitor a significant conformational change of the tRNA molecule upon interaction with the protein. However, at times long after the onset of complex formation, spectral changes indicate that the upper part of the acceptor helix becomes distorted.     

Fluorescence labeling of an aminoacyl-tRNA at the 3'-end and its interaction with elongation factor Tu.GTP

A new approach for the fluorescence labeling of an aminoacyl-tRNA at the 3'-end is applied to study its interaction with bacterial elongation factor Tu (EF-Tu) and GTP at equilibrium. The penultimate cytidine residue in yeast tRNATyr-C-C-A was replaced by 2-thiocytidine (s2C). The resulting tRNATyr-C-s2C-A was aminoacylated and then alkylated at the s2C residue with N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid (1,5-I-AEDANS). A greater than 100% increase in the intensity of fluorescence emission of the modified Tyr-tRNATyr-C-s2C(AEDANS)-A was observed upon interaction with EF-Tu.GTP. A ternary complex dissociation constant of 1.27 X 10(-8) M was calculated from this direct interaction. Using such fluorescent aminoacyl-tRNA, the affinity of any unmodified aminoacyl-tRNA can be determined by competition experiments. By this approach, we show here that the affinity of unmodified Tyr-tRNATyr-C-C-A is identical to that of the modified Tyr-tRNATyr. This indicates that the fluorescence labeling procedure applied does not alter the affinity of the aminoacyl-tRNA for EF-Tu.GTP. The introduction of 2-thiocytidine into nucleic acids and their labeling with spectroscopic reporter groups may provide a unique means of investigating various types of nucleic acid-protein interactions.     

Affinity labeling of the GDP/GTP binding site in Thermus thermophilus elongation factor Tu

Elongation factor Tu from Thermus thermophilus was treated successively with periodate-oxidized GDP or GTP and cyanoborohydride. Covalently modified cyanogen bromide or trypsin fragments of the protein were isolated, and the position of their modification was determined. Lysine residues 52 and 137 were heavily labeled, lysine-137 being considerably more reactive in the GTP form as compared to the GDP form of the protein. These residues are in the proximity of the GDP/GTP binding site. Lys-325 was also labeled, but to a lower extent. The part of the EF-Tu containing residue 52 is missing in crystallized EF-Tu.GDP from Escherichia coli [Jurnak, F. (1985) Science (Washington, D.C.) 230, 32-36]. These results place the part of T. thermophilus EF-Tu corresponding to the missing fragment in E. coli EF-Tu in the vicinity of the nucleotide binding site and allow its role in the interaction with aminoacyl-tRNA and elongation factor Ts to be evaluated. Cross-linking of EF-Tu.GDP by irradiation at 257 nm showed that a sequence of 10 amino acids residues which is found in the Thermus thermophilus elongation factor Tu but not in other homologous bacterial proteins is located in the vicinity of the GDP/GTP binding site.     

SmpB triggers GTP hydrolysis of elongation factor Tu on ribosomes by compensating for the lack of codon-anticodon interaction during trans-translation initiation

Bacterial tmRNA rescues ribosomes that stall because of defective mRNAs via the trans-translation process. Although entry of the charged transfer messenger RNA (tmRNA) into the ribosome proceeded in the absence of elongation factor (EF-Tu) and in the presence of EF-Tu and the antibiotic kirromycin, evidence was found for the involvement of EF-Tu in trans-translation initiation. The polyalanine synthesis system attained by using a tmRNA variant consisting of only the tRNA-like domain revealed that it was completely dependent on the presence of SmpB and greatly enhanced by EF-Tu and EF-G. Actually, ribosome-dependent GTPase activity of EF-Tu was stimulated by the addition of SmpB and tmRNA but independently of template mRNA, demonstrating that SmpB compensates for the lack of codon-anticodon interaction during the first step of the trans-translation initiation. Based on these results, we suggest that SmpB structurally mimics the anticodon arm of tRNA and elicits GTP hydrolysis of EF-Tu upon tmRNA accommodation in the A site of the ribosome.     

Mechanism of the chemical step for the guanosine triphosphate (GTP) hydrolysis catalyzed by elongation factor Tu

Elongation factor Tu (EF-Tu), the protein responsible for delivering aminoacyl-tRNAs (aa-tRNAs) to ribosomal A site during translation, belongs to the group of guanosine-nucleotide (GTP/GDP) binding proteins. Its active 'on'-state corresponds to the GTP-bound form, while the inactive 'off'-state corresponds to the GDP-bound form. In this work we focus on the chemical step, GTP+H(2)O-->GDP+Pi, of the hydrolysis mechanism. We apply molecular modeling tools including molecular dynamics simulations and the combined quantum mechanical-molecular mechanical calculations for estimates of reaction energy profiles for two possible arrangements of switch II regions of EF-Tu. In the first case we presumably mimic binding of the ternary complex EF-Tu.GTP.aa-tRNA to the ribosome and allow the histidine (His85) side chain of the protein to approach the reaction active site. In the second case, corresponding to the GTP hydrolysis by EF-Tu alone, the side chain of His85 stays away from the active site, and the chemical reaction GTP+H(2)O-->GDP+Pi proceeds without participation of the histidine but through water molecules. In agreement with the experimental observations which distinguish rate constants for the fast chemical reaction in EF-Tu.GTP.aa-tRNA.ribosome and the slow spontaneous GTP hydrolysis in EF-Tu, we show that the activation energy barrier for the first scenario is considerably lower compared to that of the second case.     

Essential role of histidine 84 in elongation factor Tu for the chemical step of GTP hydrolysis on the ribosome

Elongation factor Tu (EF-Tu) is a GTP-binding protein that delivers aminoacyl-tRNA to the A site of the ribosome during protein synthesis. The mechanism of GTP hydrolysis in EF-Tu on the ribosome is poorly understood. It is known that mutations of a conserved histidine residue in the switch II region of the factor, His84 in Escherichia coli EF-Tu, impair GTP hydrolysis. However, the partial reaction which is directly affected by mutations of His84 was not identified and the effect on GTP hydrolysis was not quantified. Here, we show that the replacement of His84 with Ala reduces the rate constant of GTP hydrolysis more than 10(6)-fold, whereas the preceding steps of ternary complex binding to the ribosome, codon recognition and, most importantly, the GTPase activation step are affected only slightly. These results show that His84 plays a key role in the chemical step of GTP hydrolysis. Rate constants of GTP hydrolysis by wild-type EF-Tu, measured using the slowly hydrolyzable GTP analog, GTPgammaS, showed no dependence on pH, indicating that His84 does not act as a general base. We propose that the catalytic role of His84 is to stabilize the transition state of GTP hydrolysis by hydrogen bonding to the attacking water molecule or, possibly, the gamma-phosphate group of GTP.     

EPR studies of the Mn(II) complex with elongation factor Tu and GDP Identification of oxygen ligands to Mn(II) by observation of 17O superhyperfine coupling

The coordination sphere of Mn(II) in the complex with GDP and elongation factor Tu from Escherichia coli has been probed by EPR spectroscopy with 17O-labeled ligands. Inhomogeneous broadening in the EPR signals for Mn(II) due to unresolved superhyperfine coupling to the 17O nucleus was used to identify directly bound oxygen ligands. Results with GDP selectively enriched with 17O either in the alpha-phosphate or in the beta-phosphate revealed that GDP was a beta-monodentate ligand for Mn(II) in the complex with the protein. Results with 17O-enriched water showed that two water molecules are coordinated to the Mn(II). The EPR spectrum for the complex is characteristic of octahedral coordination for Mn(II). Hence, three ligands from the protein are required to complete the sextet of ligands.     

Fluorescence characterization of the interaction of various transfer RNA species with elongation factor Tu.GTP: evidence for a new functional role for elongation factor Tu in protein biosynthesis

The ubiquity of elongation factor Tu (EF-Tu)-dependent conformational changes in amino-acyl-tRNA (aa-tRNA) and the origin of the binding energy associated with aa-tRNA.EF-Tu.GTP ternary complex formation have been examined spectroscopically. Fluorescein was attached covalently to the 4-thiouridine base at position 8 (s4U-8) in each of four elongator tRNAs (Ala, Met-m, Phe, and Val). Although the probes were chemically identical, their emission intensities in the free aa-tRNAs differed by nearly 3-fold, indicating that the dyes were in different environments and hence that the aa-tRNAs had different tertiary structures near s4U-8. Upon association with EF-Tu.GTP, the emission intensities increased by 244%, 57%, or 15% for three aa-tRNAs due to a change in tRNA conformation; the fourth aa-tRNA exhibited no fluorescence change upon binding to EF-Tu.GTP. Despite the great differences in the emission intensities of the free aa-tRNAs and in the magnitudes of their EF-Tu-dependent intensity increases, the emission intensity per aa-tRNA molecule was nearly the same (within 9% of the average) for the four aa-tRNAs when bound to EF-Tu-GTP. Thus, the binding of EF-Tu.GTP induced or selected a tRNA conformation near s4U-8 that was very similar, and possibly the same, for each aa-tRNA species. It therefore appears that EF-Tu functions, at least in part, by minimizing the conformational diversity in aa-tRNAs prior to their beginning the recognition and binding process at the single decoding site on the ribosome. Since an EF-Tu-dependent fluorescence change was also observed with fluorescein-labeled tRNA(Phe), the protein-dependent structural change is effected by direct interactions between EF-Tu and the tRNA and does not require the aminoacyl group. The Kd of the tRNA(Phe).EF-Tu.GTP ternary complex was determined, at equilibrium, to be 2.6 microM by the ability of the unacylated tRNA to compete with fluorescent Phe-tRNA for binding to the protein. Comparison of this Kd with that of the Phe-tRNA ternary complex showed that in this case the aminoacyl moiety contributed 4.3 kcal/mol toward ternary complex formation at 6 degrees C but that the bulk of the binding energy in the ternary complex was derived from direct protein-tRNA interactions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Microcalorimetric study of elongation factor Tu from Thermus thermophilus in nucleotide-free,GDP and GTP forms and in the presence of elongation factor Ts

Elongation factor (EF) Tu undergoes profound nucleotide-dependent conformational changes in its functional cycle. The thermodynamic parameters of the different Thermus thermophilus EF-Tu forms, its domains I, II/III and III, were determined by microcalorimetry. Thermal transitions of the EF-Tu.GDP and EF-Tu.guanosine-5'-[beta,gamma-imido]triphosphate have a cooperative two-state character. Nucleotide removal affected the cooperativity of the thermal transition of EF-Tu. Microcalorimetric measurements of nucleotide-free EF-Tu and its separated domains showed that domains II/III have the main stabilizing role for the whole protein. Despite the fact that strong interactions between elongation factors Tu and Ts from T. thermophilus at 20 degrees C exist, the thermal transition of neither protein in the complex was significantly affected.     

Characterization of the interaction between the nucleotide exchange factor EF-Ts from nematode mitochondria and elongation factor Tu

Caenorhabditis elegans mitochondria have two elongation factor (EF)-Tu species, denoted EF-Tu1 and EF-Tu2. Recombinant nematode EF-Ts purified from Escherichia coli bound both of these molecules and also stimulated the translational activity of EF-Tu, indicating that the nematode EF-Ts homolog is a functional EF-Ts protein of mitochondria. Complexes formed by the interaction of nematode EF-Ts with EF-Tu1 and EF-Tu2 could be detected by native gel electrophoresis and purified by gel filtration. Although the nematode mitochondrial (mt) EF-Tu molecules are extremely unstable and easily form aggregates, native gel electrophoresis and gel filtration analysis revealed that EF-Tu·EF-Ts complexes are significantly more soluble. This indicates that nematode EF-Ts can be used to stabilize homologous EF-Tu molecules for experimental purposes. The EF-Ts bound to two eubacterial EF-Tu species (E.coli and Thermus thermophilus). Although the EF-Ts did not bind to bovine mt EF-Tu, it could bind to a chimeric nematode–bovine EF-Tu molecule containing domains 1 and 2 from bovine mt EF-Tu. Thus, the nematode EF-Ts appears to have a broad specificity for EF-Tu molecules from different species.     

Mutagenesis of the active site lysine 221 of the pyruvate kinase from Bacillus stearothermophilus

Lysine 221 of the pyruvate kinase from Bacillus stearothermophilus was mutated to arginine, leucine, asparatic acid and cysteine. All the mutated enzymes were 10(4) to 10(5) times less active than the wild-type enzyme. The cysteine-free enzyme C9S/C268S, and the enzyme C9S/C268S/K221C, which possessed a unique sulfhydryl group at position 221, were prepared. The former had comparable activity to the wild-type enzyme and the latter was 10(4) times less active. These enzymes were denatured and renatured after aminoethylation. The C9S/C268S/K221C enzyme failed to regain its activity when renatured without aminoethylation; but when it was renatured after aminoethylation, it regained 4.5% of the activity of the C9S/C268S enzyme. This evidence suggests the importance of the Lys221 for the pyruvate kinase activity. The kinetic parameters of the S-aminoethylated C9S/C268S/K221C enzyme suggest that it has decreased affinity for phosphoenolpyruvate.