Isolation and characterization of Streptomyces aureofaciens protein-synthesis elongation factor Tu in an aggregated state

The ability of EF-Tu to aggregate spontaneously was employed for the purification of homogeneous EF-Tu . GDP from Streptomyces aureofaciens. The formation of filamentous structures in the aggregated EF-Tu was demonstrated in a light microscope. The purified factor, with a specific activity of 19,100 +/- 1,000 units/mg in [3H]GDP exchange, was shown to be active in the translation of poly(U). Aggregated EF-Tu . GDP exhibited almost eight-times lower GDP-exchange capacity at 2 degrees C than at 30 degrees C. This suggests that GDP-binding sites are not freely accessible at lower temperatures in the aggregated factor, in contrast to Escherichia coli polymerized EF-Tu. Turbidimetric assays revealed that the solubilization of diluted aggregated S. aureofaciens EF-Tu is strongly dependent on temperature and causes an increase in the number of accessible GDP-binding sites.     

The crystal structure of Sulfolobus solfataricus elongation factor 1alpha in complex with GDP reveals novel features in nucleotide binding and exchange

The crystal structure of elongation factor 1alpha from the archaeon Sulfolobus solfataricus in complex with GDP (SsEF-1alpha.GDP) at 1.8 A resolution is reported. As already known for the eubacterial elongation factor Tu, the SsEF-1alpha.GDP structure consists of three different structural domains. Surprisingly, the analysis of the GDP-binding site reveals that the nucleotide- protein interactions are not mediated by Mg(2+). Furthermore, the residues that usually co-ordinate Mg(2+) through water molecules in the GTP-binding proteins, though conserved in SsEF-1alpha, are located quite far from the binding site. [(3)H]GDP binding experiments confirm that Mg(2+) has only a marginal effect on the nucleotide exchange reaction of SsEF-1alpha, although essential to GTPase activity elicited by SsEF-1alpha. Finally, structural comparisons of SsEF- 1alpha.GDP with yeast EF-1alpha in complex with the nucleotide exchange factor EF-1beta shows that a dramatic rearrangement of the overall structure of EF-1alpha occurs during the nucleotide exchange.     

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.     

Acute effects of a beta-adrenoceptor agonist (BRL 26830A) on rat brown-adipose-tissue mitochondria. Increased GDP binding and GDP-sensitive proton conductance without changes in the concentration of uncoupling protein.

GDP binding, proton conductance and the specific concentration of uncoupling protein were measured in brown-adipose-tissue mitochondria of rats treated acutely with the novel beta-agonist, BRL 26830A. At 1 h after dosing with BRL 26830A, mitochondrial GDP binding was increased more than 2-fold. The increase in binding resulted from an increase in the number of binding sites. An iterative analysis of Scatchard binding data suggested that there is only one high-affinity GDP-binding site (Kd 0.3 microM) in brown-adipose-tissue mitochondria. The acute increase in GDP binding produced by treatment with BRL 26830A occurred without any alteration in the specific mitochondrial concentration of uncoupling protein, as determined by radioimmunoassay. Treatment with the beta-agonist did, however, lead to a small increase in the GDP-sensitive component of mitochondrial proton conductance. These results indicate that GDP-binding sites on uncoupling protein can be rapidly unmasked after treatment with a brown-fat-specific beta-agonist, and that the increase in binding reflects an increase in the activity of the mitochondrial proton-conductance pathway.     

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.     

Conformational change of elongation factor Tu (EF-Tu) induced by antibiotic binding. Crystal structure of the complex between EF-Tu.GDP and aurodox

Aurodox is a member of the family of kirromycin antibiotics, which inhibit protein biosynthesis by binding to elongation factor Tu (EF-Tu). We have determined the crystal structure of the 1:1:1 complex of Thermus thermophilus EF-Tu with GDP and aurodox to 2.0-A resolution. During its catalytic cycle, EF-Tu adopts two strikingly different conformations depending on the nucleotide bound: the GDP form and the GTP form. In the present structure, a GTP complex-like conformation of EF-Tu is observed, although GDP is bound to the nucleotide-binding site. This is consistent with previous proposals that aurodox fixes EF-Tu on the ribosome by locking it in its GTP form. Binding of EF-Tu.GDP to aminoacyl-tRNA and mutually exclusive binding of kirromycin and elongation factor Ts to EF-Tu can be explained on the basis of the structure. For many previously observed mutations that provide resistance to kirromycin, it can now be understood how they prevent interaction with the antibiotic. An unexpected feature of the structure is the reorientation of the His-85 side chain toward the nucleotide-binding site. We propose that this residue stabilizes the transition state of GTP hydrolysis, explaining the acceleration of the reaction by kirromycin-type antibiotics.     

Evidence that the acute unmasking of GDP-binding sites in brown adipose tissue mitochondria is not dependent on mitochondrial swelling

The role of mitochondrial swelling in the unmasking of GDP-binding sites on brown adipose tissue mitochondria has been examined in mice. Acute cold exposure (6 degrees C for 1 h) led to increases in GDP binding without changes in the concentration of uncoupling protein, indicating that an unmasking of binding sites had occurred. Measurements of mitochondrial matrix volume suggested that an acute unmasking of GDP-binding sites took place without swelling of the mitochondria. In addition, the induction of a rapid preswelling of the mitochondria by incubation in KCl in the presence of valinomycin did not affect the cold-induced unmasking of GDP-binding sites. It is concluded that the acute unmasking of GDP-binding sites on uncoupling protein in brown adipose tissue is not due simply to mitochondrial swelling.     

Interaction of mammalian mitochondrial elongation factor EF-Tu with guanine nucleotides

Elongation factor Tu (EF-Tu) promotes the binding of aminoacyl-tRNA (aa-tRNA) to the acceptor site of the ribosome. During the elongation cycle, EF-Tu interacts with guanine nucleotides, aa-tRNA and its nucleotide exchange factor (EF-Ts). Quantitative determination of the equilibrium dissociation constants that govern the interactions of mammalian mitochondrial EF-Tu (EF-Tu(mt)) with guanine nucleotides was the focus of the work reported here. Equilibrium dialysis with [3H]GDP was used to measure the equilibrium dissociation constant of the EF-Tu(mt) x GDP complex (K(GDP) = 1.0 +/- 0.1 microM). Competition of GTP with a fluorescent derivative of GDP (mantGDP) for binding to EF-Tu(mt) was used to measure the dissociation constant of the EF-Tu(mt) x GTP complex (K(GTP) = 18 +/- 9 microM). The analysis of these data required information on the dissociation constant of the EF-Tu(mt) x mantGDP complex (K(mGDP) = 2.0 +/- 0.5 microM), which was measured by equilibrium dialysis. Both K(GDP) and K(GTP) for EF-Tu(mt) are quite different (about two orders of magnitude higher) than the dissociation constants of the corresponding complexes formed by Escherichia coli EF-Tu. The forward and reverse rate constants for the association and dissociation of the EF-Tu(mt) x GDP complex were determined using the change in the fluorescence of mantGDP upon interaction with EF-Tu(mt). These values are in agreement with a simple equilibrium binding interaction between EF-Tu(mt) and GDP. The results obtained are discussed in terms of the recently described crystal structure of the EF-Tu(mt) x GDP complex.     

Crystal structure and functional analysis of the eukaryotic class II release factor eRF3 from S. pombe

Translation termination in eukaryotes is governed by two interacting release factors, eRF1 and eRF3. The crystal structure of the eEF1alpha-like region of eRF3 from S. pombe determined in three states (free protein, GDP-, and GTP-bound forms) reveals an overall structure that is similar to EF-Tu, although with quite different domain arrangements. In contrast to EF-Tu, GDP/GTP binding to eRF3c does not induce dramatic conformational changes, and Mg(2+) is not required for GDP binding to eRF3c. Mg(2+) at higher concentration accelerates GDP release, suggesting a novel mechanism for nucleotide exchange on eRF3 from that of other GTPases. Mapping sequence conservation onto the molecular surface, combined with mutagenesis analysis, identified the eRF1 binding region, and revealed an essential function for the C terminus of eRF3. The N-terminal extension, rich in acidic amino acids, blocks the proposed eRF1 binding site, potentially regulating eRF1 binding to eRF3 in a competitive manner.     

Isolation,crystallisation, and preliminary X-ray analysis of the bovine mitochondrial EF-Tu:GDP and EF-Tu:EF-Ts complexes

Previous studies have shown that when bovine mitochondrial elongation factor Ts (EF-Ts) is expressed in Escherichia coli, it forms a tightly associated complex with E. coli elongation factor Tu (EF-Tu). In contrast to earlier experiments, purification of free mitochondrial EF-Ts was accomplished under nondenaturing conditions since only about 60% of the expressed EF-Ts copurified with E. coli EF-Tu. The bovine mitochondrial EF-Tu:GDP complex, the homologous mitochondrial EF-Tu:EF-Ts complex, and the heterologous E. coli/mitochondrial EF-Tu:EF-Ts complex were isolated and crystallised. The crystals of the EF-Tu:GDP complex diffract to 1.94 A and belong to space group P2(1) with cell parameters a=59.09 A, b=119.78 A, c=128.89 A and beta=96.978 degrees. The crystals of the homologous mitochondrial EF-Tu:EF-Ts complex diffract to 4 A and belong to space group C2 with cell parameters a=157.7 A, b=151.9 A, c=156.9 A, and beta=108.96 degrees.     

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.     

The elongation factor Tu.kirromycin complex has two binding sites for tRNA molecules

The interaction of the polypeptide chain elongation factor Tu (EF-Tu) with the antibiotic kirromycin and tRNA has been studied by measuring the extent of protein modification with N-tosyl-L-phenylalanine chloromethylketone (TPCK) and N-ethylmaleimide (NEM). Kirromycin protects both EF-Tu.GDP and EF-Tu.GTP against modification with TPCK. Binding of aminoacyl-tRNA added at increasing concentrations to a solution of 40 microM EF-Tu.GDP.kirromycin complex re-exposes the TPCK target site on the protein. However, when the aminoacyl-tRNA concentration is raised beyond 20 microM, TPCK labeling drops again and is blocked completely at approximately 300 microM aminoacyl-tRNA. By contrast, addition of uncharged tRNA or N- acetylaminoacyl -tRNA enhances TPCK labeling of the protein over the entire tRNA concentration range studied. These data strongly suggest that kirromycin induces in EF-Tu.GDP an additional tRNA binding site that can bind uncharged tRNA, aminoacyl-tRNA, and N- acetylaminoacyl -tRNA. Support for this assumption is provided by measuring the modification of EF-Tu.GDP with the sulfhydryl reagent NEM. Moreover, NEM modification also indicates an additional tRNA binding site on EF-Tu.GTP.kirromycin, which could not be detected with TPCK. Mapping of the tryptic peptides of EF-Tu.GDP labeled with [14C]TPCK revealed only one target site for this agent, i.e., cysteine-81. Modification occurred at the same site in the presence and in the absence of kirromycin and uncharged tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of Saccharomyces cerevisiae mitochondrial elongation factor Tu

Yeast mitochondrial elongation factor Tu (EF-Tu) was purified 200-fold from a mitochondrial extract of Saccharomyces cerevisiae to yield a single polypeptide of Mr = approximately 47,000. The factor was detected by complementation with Escherichia coli elongation factor G and ribosomes in an in vitro phenylalanine polymerization reaction. Mitochondrial EF-Tu, like E. coli EF-Tu, catalyzes the binding of aminoacyl-tRNA to ribosomes and possesses an intrinsic GTP hydrolyzing activity which can be activated either by kirromycin or by ribosomes. Kinetic and binding analyses of the interactions of mitochondrial EF-Tu with guanine nucleotides yielded affinity constants for GTP and GDP of approximately 5 and 25 microM, respectively. The corresponding affinity constants for the E. coli factor are approximately 0.3 and 0.003 microM, respectively. In keeping with these observations, we found that purified mitochondrial EF-Tu, unlike E. coli EF-Tu, does not contain endogenously bound nucleotide and is not stabilized by GDP. In addition, we have been unable to detect a functional counterpart to E. coli EF-Ts in extracts of yeast mitochondria and E. coli EF-Ts did not detectably stimulate amino acid polymerization with mitochondrial EF-Tu or enhance the binding of guanine nucleotides to the factor. We conclude that while yeast mitochondrial EF-Tu is functionally analogous to and interchangeable with E. coli EF-Tu, its affinity for guanine nucleotides and interaction with EF-Ts are quite different from those of E. coli EF-Tu.     

Generation of potential structures for the G-domain of chloroplast EF-Tu using comparative molecular modeling

Comparative molecular modeling has been used to generate several possible structures for the G-domain of chloroplast elongation factor Tu (EF-Tu(chl)) based on the crystallographic data of the homologous E. coli protein. EF-Tu(chl) contains a 10 amino acid insertion not present in the E. coli protein and this region has been modeled based on its predicted secondary structure. The insertion appears to lie on the surface of the protein. Its orientation could not be determined unequivocally but several likely structures for the nucleotide binding domain of EF-Tu(chl) have been developed. The effects of the presence of water in the Mg2+ coordination sphere and of the protonation state of the GDP ligand on the conformation of the guanine nucleotide binding site have been examined. Relative binding constants of several guanine nucleotide analogs for EF-Tu(chl) have been obtained. The interactions between EF-Tu(chl) and GDP predicted to be important by the models that have been developed are discussed in relation to the nucleotide binding properties of this factor and to the interactions proposed to be important in the binding of guanine nucleotides to related proteins.     

Properties of the elongation factor 1 alpha in the thermoacidophilic archaebacterium Sulfolobus solfataricus

The elongation factor 1 alpha (aEF-1 alpha) was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus by chromatographic procedures utilising DEAE-Sepharose, hydroxyapatite and FPLC on Mono S. The purified protein binds [3H]GDP at a 1:1 molar ratio and it is essential for poly(Phe) synthesis in vitro; it also binds GTP but not ATP. These findings indicate that aEF-1 alpha is the counterpart of the eubacterial elongation factor Tu (EF-Tu). Purified aEF-1 alpha is a monomeric protein with a relative molecular mass of 49,000 as determined by SDS/PAGE and by gel filtration on Sephadex G-100; its isoelectric point is 9.1. The overall amino acid composition did not reveal significant differences when compared with the amino acid composition of eubacterial EF-Tu from either Escherichia coli or Thermus thermophilus, of eukaryotic EF-1 alpha from Artemia salina or of archaebacterial EF-1 alpha from Methanococcus vannielii. The close similarities between the average hydrophobicity and the numbers of hydrogen-bond-forming or non-helix-forming residues suggest that common structural features exist among the factors compared. aEF-1 alpha shows remarkable thermophilic properties, as demonstrated by the rate of [3H]GDP binding which increases with temperature, reaching a maximum at 95 degrees C; it is also quite heat-resistant, since after a 6-h exposure at 60 degrees C and 87 degrees C the residual [3H]GDP-binding ability was still 90% and 54% of the control, respectively. The affinity of aEF-1 alpha for GDP and GTP was also evaluated. At 80 degrees C Ka' for GDP was about 30-fold higher than Ka' for GTP; at the same temperature Kd' for GDP was 1.7 microM and Kd' for GTP was 50 microM; these values were 300-fold and 100-fold higher, respectively, than those reported for E. coli EF-Tu at 30 degrees C; compared to the values at 0 degree C of EF-Tu from E. coli and T. thermophilus or EF-1 alpha from A. salina, pig liver and calf brain, smaller differences were observed with eukaryotic factors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction of mitochondrial elongation factor Tu with aminoacyl-tRNA and elongation factor Ts

Elongation factor (EF) Tu promotes the binding of aminoacyl-tRNA (aa-tRNA) to the acceptor site of the ribosome. This process requires the formation of a ternary complex (EF-Tu.GTP.aa-tRNA). EF-Tu is released from the ribosome as an EF-Tu.GDP complex. Exchange of GDP for GTP is carried out through the formation of a complex with EF-Ts (EF-Tu.Ts). Mammalian mitochondrial EF-Tu (EF-Tu(mt)) differs from the corresponding prokaryotic factors in having a much lower affinity for guanine nucleotides. To further understand the EF-Tu(mt) subcycle, the dissociation constants for the release of aa-tRNA from the ternary complex (K(tRNA)) and for the dissociation of the EF-Tu.Ts(mt) complex (K(Ts)) were investigated. The equilibrium dissociation constant for the ternary complex was 18 +/- 4 nm, which is close to that observed in the prokaryotic system. The kinetic dissociation rate constant for the ternary complex was 7.3 x 10(-)(4) s(-)(1), which is essentially equivalent to that observed for the ternary complex in Escherichia coli. The binding of EF-Tu(mt) to EF-Ts(mt) is mutually exclusive with the formation of the ternary complex. K(Ts) was determined by quantifying the effects of increasing concentrations of EF-Ts(mt) on the amount of ternary complex formed with EF-Tu(mt). The value obtained for K(Ts) (5.5 +/- 1.3 nm) is comparable to the value of K(tRNA).     

Enacyloxin IIa pinpoints a binding pocket of elongation factor Tu for development of novel antibiotics

Elongation factor (EF-) Tu.GTP is the carrier of aminoacyl-tRNA to the programmed ribosome. Enacyloxin IIa inhibits bacterial protein synthesis by hindering the release of EF-Tu.GDP from the ribosome. The crystal structure of the Escherichia coli EF-Tu.guanylyl iminodiphosphate (GDPNP).enacyloxin IIa complex at 2.3 A resolution presented here reveals the location of the antibiotic at the interface of domains 1 and 3. The binding site overlaps that of kirromycin, an antibiotic with a structure that is unrelated to enacyloxin IIa but that also inhibits EF-Tu.GDP release. As one of the major differences, the enacyloxin IIa tail borders a hydrophobic pocket that is occupied by the longer tail of kirromycin, explaining the higher binding affinity of the latter. EF-Tu.GDPNP.enacyloxin IIa shows a disordered effector region that in the Phe-tRNAPhe.EF-Tu (Thermus aquaticus).GDPNP.enacyloxin IIa complex, solved at 3.1 A resolution, is stabilized by the interaction with tRNA. This work clarifies the structural background of the action of enacyloxin IIa and compares its properties with those of kirromycin, opening new perspectives for structure-guided design of novel antibiotics.     

Refined structure of elongation factor EF-Tu from Escherichia coli.

The crystal structure of trypsin-modified elongation factor Tu from Escherichia coli, in complex with the cofactor guanosine diphosphate has been refined to a crystallographic R-factor of 19.3%, at 2.6 A resolution. In the model described, the root-mean-square deviation from ideality is 0.019 A for bond distances and 3.9 degrees for angles. The protein consists of three domains: an alpha/beta domain (residues 1 to 200), containing the binding site of the GDP cofactor, and consisting of a six-stranded beta-pleated sheet, six alpha-helices, and two all-beta domains (residues 209 to 299 and 300 to 393), belonging to the tertiary structural class of antiparallel beta-barrels. The GDP-binding domain has a folding that is found in other GDP-binding proteins. Elongation factor Tu interacts with proteins, nucleic acids and nucleotides, making this molecule well suited as a model system for the study of these interactions.     

Structure of an EF-Tu complex with a thiazolyl peptide antibiotic determined at 2.35 A resolution: atomic basis for GE2270A inhibition of EF-Tu

The structure of a 1:1 molar complex between Escherichia coli elongation factor (EF) Tu-GDP and the cyclic thiazolyl peptide antibiotic, GE2270A, has been determined by X-ray diffraction analysis to a resolution of 2.35 A and refined to a crystallographic refinement factor of 20.6%. The antibiotic binds in the second domain of EF-Tu-GDP, making contact with three segments of amino acids (residues 215-230, 256-264, and 273-277). The majority of the protein-antibiotic contacts are van der Waals interactions. A striking feature of the antibiotic binding site is the presence of a salt bridge, not previously observed in other EF-Tu complexes. The ionic interaction between Arg 223 and Glu 259 forms over the antibiotic and probably accounts for the strong affinity observed between EF-Tu and GE2270A. Arg 223 and Glu 259 are highly conserved, but not invariant throughout the prokaryotic EF-Tu family, suggesting that the antibiotic may bind EF-Tu from some organisms better than others may. Superposition of the antibiotic binding site on the EF-Tu-GTP conformation reveals that one region of the antibiotic would form steric clashes with the guanine nucleotide-binding domain in the GTP, but not the GDP, conformation. Another region of the antibiotic binds to the same site as the aminoacyl group of tRNA. Together with prior biochemical studies, the structural findings confirm that GE2270A inhibits protein synthesis by blocking the GDP to GTP conformational change and by directly competing with aminoacyl-tRNA for the same binding site on EF-Tu. In each of the bacterial strains that are resistant to GE2270A, the effect of a site-specific mutation in EF-Tu could explain resistance. Comparison of the GE2270A site in EF-Tu with sequence homologues, EF-G and EF-1alpha, suggests steric clashes that would prevent the antibiotic from binding to translocation factors or to the eukaryotic equivalent of EF-Tu. Although GE2270A is a potent antibiotic, its clinical efficacy is limited by its low aqueous solubility. The results presented here provide the details necessary to enhance the solubility of GE2270A without disrupting its inhibitory properties.