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.     

Bovine mitochondrial protein synthesis elongation factors. Identification and initial characterization of an elongation factor Tu-elongation factor Ts complex

Animal mitochondrial protein synthesis factors elongation factor (EF) Tu and EF-Ts have been purified as an EF-Tu.Ts complex from crude extracts of bovine liver mitochondria. The mitochondrial complex has been purified 10,000-fold to near homogeneity by a combination of chromatographic procedures including high performance liquid chromatography. The mitochondrial EF-Tu.Ts complex is very stable and cannot be dissociated even in the presence of high concentrations of guanine nucleotides. No guanine nucleotide binding to this complex can be observed in the standard nitrocellulose filter binding assay. Mitochondrial EF-Ts activity can be detected by its ability to facilitate guanine nucleotide exchange with Escherichia coli EF-Tu. The EF-Tumt exhibits similar levels of activity on isolated mammalian mitochondrial and E. coli ribosomes, but displays minimal activity on Euglena gracilis chloroplast 70 S ribosomes and has no detectable activity on wheat germ cytoplasmic ribosomes. In contrast to the bacterial EF-Tu and the EF-Tu from the chloroplast of E. gracilis, the ability of the mitochondrial factor to catalyze polymerization is not inhibited by the antibiotic kirromycin.     

Spin-labelled analogues of GDP and GTP as site-specific reporter groups for guanosine nucleotide-binding proteins

New derivatives of GDP and GTP have been synthesized for the spectroscopic investigation of the interaction between guanosine nucleotides and guanosine nucleotide-binding proteins. The 3'-hydroxyl group in these nucleotides was replaced by a 3'-amino group, which was further derivatized by the introduction of a spin-label reporter group. The biological activity of 3'SL-GDP and 3'SL-GTP could be demonstrated by measuring the interaction of these spin-labelled derivatives with bacterial elongation factor Tu. The amino modification and spin labelling only slightly influenced the affinity of the guanosine nucleotides for EF-Tu from Escherichia coli or Thermus thermophilus. Electron paramagnetic resonance (EPR) measurements revealed a strong immobilization of the labelled nucleotides upon binding to T. thermophilus EF-Tu. Significant differences between the spectra of EF-Tu X 3'SL-GDP, EF-Tu X 3'SL-GTP and aminoacyl-tRNA X EF-Tu X 3'SL-GTP ternary complexes were observed. Our data demonstrate that spin-labelled guanosine nucleotides can be used as sensitive spectroscopic probes for the investigation of the local environment of the nucleotide-binding site during distinct functional states of a guanosine nucleotide-binding protein.     

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.     

Stimulation of Escherichia coli adenylate cyclase activity by elongation factor Tu, a GTP-binding protein essential for protein synthesis

A unique feature of eucaryotic adenylate cyclases is their interaction with GTP-binding proteins that mediate hormonal responses. Until now, there has been no evidence for regulation of Escherichia coli adenylate cyclase by a GTP-binding protein. We describe here that the most abundant protein in E. coli, the GTP-binding protein EF-Tu, which is important as an elongation factor in protein synthesis, also serves as a stimulator of adenylate cyclase activity. Homogeneous EF-Tu specifically increased the activity of purified adenylate cyclase as much as 70%; other E. coli GTP-binding proteins had no effect on enzyme activity. A study of the guanine nucleotide specificity for EF-Tu-mediated stimulation of adenylate cyclase activity suggested that the preferred activator is EF-Tu X GDP. To account for the GTP-specific stimulation of adenylate cyclase activity observed in intact cells, we propose that the nucleotide specificity for EF-Tu-dependent activation of adenylate cyclase is governed by other factors in the cell.     

Structure-function relationships of elongation factor Tu as studied by mutagenesis.

We have modified elongation factor Tu (EF-Tu) from Escherichia coli via mutagenesis of its encoding tufA gene to study its function-structure relationships. The isolation of the N-terminal half molecule of EF-Tu (G domain) has facilitated the analysis of the basic EF-Tu activities, since the G domain binds the substrate GTP/GDP, catalyzes the GTP hydrolysis and is not exposed to the allosteric constraints of the intact molecule. So far, the best studied region has been the guanine nucleotide-binding pocket defined by the consensus elements typical for the GTP-binding proteins. In this area most substitutions were carried out in the G domain and were found to influence GTP hydrolysis. In particular, the mutation VG20 (in both G domain and EF-Tu) decreases this activity and enhances the GDP to GTP exchange; PT82 induces autophosphorylation of Thr82 and HG84 strongly affects the GTPase without altering the interaction with the substrate. SD173, a residue interacting with (O)6 of the guanine, abolishes the GTP and GDP binding activity. Substitution of residues Gln114 and Glu117, located in the proximity of the GTP binding pocket, influences respectively the GTPase and the stability of the G domain, whereas the double replacement VD88/LK121, located on alpha-helices bordering the GTP-binding pocket, moderately reduces the stability of the G domain without greatly affecting GTPase and interaction with GTP(GDP). Concerning the effect of ligands, EF-TuVG20 supports a lower poly(Phe) synthesis but is more accurate than wild-type EF-Tu, probably due to a longer pausing on the ribosome.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Mutagenesis of bacterial elongation factor Tu at lysine 136. A conserved amino acid in GTP regulatory proteins

We have studied the effects of specific amino acid replacements in EF-Tu upon the protein's interactions with guanine nucleotides and elongation factor Ts (EFTs). We found that alterations at the lysine residue of the Asn-Lys-Cys-Asp sequence, the guanine ring-binding sequence, differentially affect the protein's ability to bind guanine nucleotides. Wild type EF-Tu (Lys-136) binds GDP and GTP much more tightly than do many of the altered proteins. Replacing lysine by arginine lowers the protein's affinity for GDP by about 20-fold relative to the change in its affinity for EF-Ts. Substitutions at residue 136 by glutamine (K136Q) and glutamic acid (K136E) further lower the protein relative affinity for GDP by factors of about 4 and 10, respectively. In contrast, replacement of the residue by isoleucine (K136I) eliminates guanine nucleotide binding as well as EF-Ts binding. Apparently, the distortion of this loop by substitution at residue 136 of a bulky hydrophobic residue can hamper the binding for both substrates or disrupt the folding of the protein. All altered proteins except EF-Tu(K136I) are able to bind tRNA(Phe); however, they require much higher concentrations of GTP than wild type EF-Tu. In minimal media, Escherichia coli cells harboring plasmids encoding EF-Tu(K136E) or EF-Tu(K136Q) suffer growth retardation relative to cells bearing the same plasmid encoding wild type EF-Tu. Co-transformation of these cells with a compatible plasmid bearing the EF-Ts gene reverses this growth problem. The growth retardation effect of some of the altered proteins can be explained by their sequestering EF-Ts. These results indicate that EF-Ts is essential to the growth of E. coli and suggest a technique for studying EF-Ts mutants as well as for identifying other guanine nucleotide exchange enzymes.     

Studies on polypeptide-chain-elongation factors from an extreme thermophile, Thermus thermophilus HB8. 2. Catalytic properties.

Catalytic properties of the elongation factors from Thermus thermophilus HB8 have been studied and compared with those of the factors from Escherichia coli. 1. The formation of a ternary guanine-nucleotide . EF-Tu . EF-Ts complex was demonstrated by gel filtration of the T. thermophilus EF-Tu . EF-Ts complex on a Sephadex G-150 column equilibrated with guanine nucleotide. The occurrence of this type of complex has not yet been proved with the factors from E. coli. 2. The dissociation constants for the complexes of T. thermophilus EF-Tu . EF-Ts with GDP and GTP were 6.1 x 10(-7) M and 1.9 x 10(-6) M respectively. On the other hand, T. thermophilus EF-Tu interacted with GDP and GTP with dissociation constants of 1.1 x 10(-9) M and 5.8 x 10(-8) M respectively. This suggests that the association of EF-Ts with EF-Tu lowered the affinity of EF-Tu for GDP by a factor of about 600 and facilitated the nucleotide exchange reaction. 3. Although the T. thermophilus EF-Tu . EF-Ts complex hardly dissociates into EF-Tu and EF-Ts, a rapid exchange was observed between free EF-Ts and the EF-Tu . EF-Ts complex using 3H-labelled EF-Ts. The exchange reaction was independent on the presence or absence of guanine nucleotides. 4. Based on the above findings, an improved reaction mechanism for the regeneration of EF-Tu . GTP from EF-Tu . GDP is proposed. 5. Studies on the functional interchangeability of EF-Tu and EF-Ts between T. thermophilus and E. coli has revealed that the factors function much more efficiently in the homologous than in the heterologous combination. 6. T. thermophilus EF-Ts could bind E. coli EF-Tu to form an EF-Tu (E. coli) . EF-Ts (T. thermophilus hybrid complex. The complex was found to exist in a dimeric form indicating that the property to form a dimer is attributable to T. thermophilus EF-Ts. On the other hand, no stable complex between E. coli EF-Ts and T. thermophilus EF-Tu has been isolated. 7. The uncoupled GTPase activity of T. thermophilus EF-G was much lower than that of E. coli EF-G. T. thermophilus EF-G formed a relatively stable binary EF-G . GDP complex, which could be isolated on a nitrocellulose membrane filter. The Kd values for EF-G . GDP and EF-G . GTP were 6.7 x 10(-7) M and 1.2 x 10(-5) M respectively. The ternary T. thermophilus EF-G . GDP . ribosome complex was again very stable and could be isolated in the absence of fusidic acid. The stability of the latter complex is probably the cause of the low uncoupled GTPase activity of T. thermophilus EF-G.     

Spectroscopic and hydrodynamic studies reveal structural differences in normal and transforming H-ras gene products

We have recorded the circular dichroism spectra of the cellular and the viral H-ras gene products both in the absence and in the presence of guanine nucleotides and analyzed these spectra in terms of the secondary structure composition of these proteins. It is shown that the GTP complex of the ras proteins has a different secondary structure composition than the GDP complex and, furthermore, that there are differences in the secondary structure of the viral ras protein and the cellular ras protein. We have also recorded and analyzed the circular dichroism spectrum of the isolated guanine nucleotide binding domain of the Escherichia coli elongation factor Tu (EF-Tu), which has been considered as a model for the tertiary structure of the ras proteins [McCormick, F., Clark, B. F. C., LaCour, T. F. M., Kjeldgaard, M., Norskov-Lauritsen, L., & Nyborg, J. (1985) Science (Washington, D.C.) 230, 78-82]. Our data show that the guanine nucleotide binding domain of EF-Tu (30% alpha-helix and 16% beta-pleated sheet for the GDP complex) has quite a different secondary structure composition than the ras proteins (e.g., the cellular ras protein has 47% alpha-helix and 22% beta-pleated sheet for the GDP complex), indicating that the protein core comprising the guanine nucleotide binding site might be similar but that major structural differences must exist at the portion outside this core. Normal and transforming ras proteins also differ slightly in their hydrodynamic properties as shown by sedimentation velocity runs in the analytical ultracentrifuge.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recognition of the universally conserved 3'-CCA end of tRNA by elongation factor EF-Tu.

Escherichia coli tRNA(Val) with pyrimidine substitutions for the universally conserved 3'-terminal adenine can be readily aminoacylated. It cannot, however, transfer valine into polypeptides. Conversely, despite being a poor substrate for valyl-tRNA synthetase, tRNA(Val) with a 3'-terminal guanine is active in in vitro polypeptide synthesis. To better understand the function of the 3'-CCA sequence of tRNA in protein synthesis, the effects of systematically varying all three bases on formation of the Val-tRNA(Val):EF-Tu:GTP ternary complex were investigated. Substitutions at C74 and C75 have no significant effect, but replacing A76 with pyrimidines decreases the affinity of valyl-tRNA(Val) for EF-Tu:GTP, thus explaining the inability of these tRNA(Val) variants to function in polypeptide synthesis. Valyl-tRNA(Val) terminating in 3'-guanine is readily recognized by EF-TU:GTP. Dissociation constants of the EF-Tu:GTP ternary complexes with valine tRNAs having nucleotide substitutions at the 3' end increase in the order adenine < guanine < uracil; EF-Tu has very little affinity for tRNA terminating in 3' cytosine. Similar observations were made in studies of the interaction of 3' end mutants of E. coli tRNA(Ala) and tRNA(Phe) with EF-Tu:GTP. These results indicate that EF-Tu:GTP preferentially recognizes purines and discriminates against pyrimidines, especially cytosine, at the 3' end of aminoacyl-tRNAs.     

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.     

Qbeta-phage resistance by deletion of the coiled-coil motif in elongation factor Ts

Elongation factor Ts (EF-Ts) is the guanine-nucleotide exchange factor of elongation factor Tu (EF-Tu), which promotes the binding of aminoacyl-tRNA to the mRNA-programmed ribosome in prokaryotes. The EF-Tu.EF-Ts complex, one of the EF-Tu complexes during protein synthesis, is also a component of RNA-dependent RNA polymerases like the polymerase from coliphage Qbeta. The present study shows that the Escherichia coli mutant GRd.tsf lacking the coiled-coil motif of EF-Ts is completely resistant to phage Qbeta and that Qbeta-polymerase complex formation is not observed. GRd.tsf is the first E. coli mutant ever described that is unable to form a Qbeta-polymerase complex while still maintaining an almost normal growth behavior. The phage resistance correlates with an observed instability of the mutant EF-Tu.EF-Ts complex in the presence of guanine nucleotides. Thus, the mutant EF-Tu.EF-Ts is the first EF-Tu.EF-Ts complex ever described that is completely inactive in the Qbeta-polymerase complex despite its almost full activity in protein synthesis. We propose that the role of EF-Ts in the Qbeta-polymerase complex is to control and trap EF-Tu in a stable conformation with affinity for RNA templates while unable to bind aminoacyl-tRNA.     

Psychrophilic elongation factor Tu from the antarctic Moraxella sp. Tac II 25: biochemical characterization and cloning of the encoding gene

The elongation factor Tu was isolated from a psychrophilic eubacterial Antarctic Moraxella strain (MoEF-Tu) and its molecular and functional properties were determined. It catalyzed the synthesis of poly(Phe) and bound specifically guanine nucleotides with an affinity for GDP about 12-fold higher than that for GTP. The affinity toward guanine nucleotides was lower than that of other eubacterial EF-Tu. The intrinsic GTPase activity of MoEF-Tu was hardly detectable but was accelerated by 2 orders of magnitude in the presence of the antibiotic kirromycin (GTPase(k)). Such a property resembled Escherichia coli EF-Tu (EcEF-Tu) even though the affinity of MoEF-Tu for the antibiotic was lower. MoEF-Tu showed a thermophilicity higher than that of EcEF-Tu; its temperature for half-denaturation was 44 degrees C. The MoEF-Tu encoding gene corresponding to E. coli tufA was cloned and sequenced. The translated protein had a calculated molecular weight of 43 288 and contained the GTP-binding sequence motifs. Concerning its primary structure, MoEF-Tu showed sequence identity with E. coli and Thermus thermophilus EF-Tu equal to 84% and 74%, respectively, while the identity with EF-1 alpha from the archaeon Sulfolobus solfataricus was equal to 32%.     

The 51-63 base pair of tRNA confers specificity for binding by EF-Tu

Elongation factor Tu (EF-Tu) exhibits significant specificity for the different elongator tRNA bodies in order to offset its variable affinity to the esterified amino acid. Three X-ray cocrystal structures reveal that while most of the contacts with the protein involve the phosphodiester backbone of tRNA, a single hydrogen bond is observed between the Glu390 and the amino group of a guanine in the 51-63 base pair in the T-stem of tRNA. Here we show that the Glu390Ala mutation of Thermus thermophilus EF-Tu selectively destabilizes binding of those tRNAs containing a guanine at either position 51 or 63 and that mutagenesis of the 51-63 base pair in several tRNAs modulates their binding affinities to EF-Tu. A comparison of Escherichia coli tRNA sequences suggests that this specificity mechanism is conserved across the bacterial domain. While this contact is an important specificity determinant, it is clear that others remain to be identified.     

Sequence of the tufA gene encoding elongation factor EF-Tu from Thermus aquaticus and overproduction of the protein in Escherichia coli.

The sequence of the tufA gene from the extreme thermophilic eubacterium Thermus aquaticus EP 00276 was determined. The GC content in third positions of codons is 89.5%, with an unusual predominance of guanosine (60.7%). The derived protein sequence differs from tufA- and tufB-encoded sequences for elongation factor Tu (EF-Tu) of Thermus thermophilus HB8, another member of the genus Thermus, in 10 of the 405 amino acid residues. Three exchanges are located in the additional loop of ten amino acids (182-191). The loop, probably involved in nucleotide binding, is absent in EF-Tu of the mesophile Escherichia coli. Since EF-Tu from E. coli is quite unstable, the protein is well-suited for analyzing molecular changes that lead to thermostabilization. Comparison of the EF-Tu domain I from E. coli and Thermus strains revealed clustered amino acid exchanges in the C-terminal part of the first helix and in adjacent residues of the second loop inferred to interact with the ribosome. Most other exchanges in the guanine nucleotide binding domain are located in loops or nearest vicinity of loops suggesting their importance for thermostability. The T. aquaticus EF-Tu was overproduced in E. coli using the tac expression system. Identity of the recombinant T. aquaticus EF-Tu was verified by Western blot analysis, N-terminal sequencing and GDP binding assays.     

Mutation of Thr445 and Ile500 of initiation factor 2 G-domain affects Escherichia coli growth rate at low temperature

The Escherichia coli protein synthesis initiation factor IF2 is a member of the large family of G-proteins. Along with translational elongation factors EF-Tu and EF-G and translational release factor RF-3, IF2 belongs to the subgroup of G-proteins that are part of the prokaryotic translational apparatus. The roles of IF2 and EF-Tu are similar: both promote binding of an aminoacyl-tRNA to the ribosome and hydrolyze GTP. In order to investigate the differences and similarities between EF-Tu and IF2 we have created point mutations in the G-domain of IF2, Thr445 to Cys, Ile500 to Cys, and the double mutation. Threonine 445 (X1), which corresponds to cysteine 81 in EF-Tu, is well conserved in the DX1X2GH consensus sequence that has been proposed to interact with GTP. The NKXD motif, in which X is isoleucine 500 in IF2, corresponds to cysteine 137 in EF-Tu, and is responsible for the binding of the guanine ring. The recombinant mutant proteins were expressed and tested in vivo for their ability to sustain growth of an Escherichia coli strain lacking the chromosomal copy of the infB gene coding for IF2. All mutated proteins resulted in cell viability when grown at 42 degrees C or 37 degrees C. However, Thr445 to Cys mutant showed a significant decrease in the growth rate at 25 degrees C. The mutant proteins were overexpressed and purified. As observed in vivo, a reduced activity at low temperature was measured when carrying out in vitro ribosome dependent GTPase and stimulation of ribosomal fMet-tRNAfMet binding.     

Activation of cholera toxin and Escherichia coli heat-labile enterotoxins by ADP-ribosylation factors, a family of 20 kDa guanine nucleotide-binding proteins

Cholera toxin and Escherichia coli heat-labile enterotoxins are responsible, in part, for the symptomatology of cholera and traveller's diarrhoea, respectively. Effects of the toxins result from ADP-ribosylation of regulatory guanine nucleotide-binding (G) proteins; the ADP-ribosylated G protein is stabilized in an activated state, resulting in prolonged effects on its target. Toxin-catalysed ADP-ribosylation is stimulated in vitro by a family of guanine nucleotide-binding proteins, c. 20 kDa, termed ADP-ribosylation factors or ARFs. In the presence of GTP, but not GDP or adenine analogues, ARFs serve as allosteric activators of the toxin. The effects are amplified by certain phospholipids and detergents which promote guanine nucleotide binding. Six different mammalian ARF genes have been identified. They encode highly conserved, ubiquitous proteins of 175 to 181 amino acids, containing consensus domains responsible for guanine nucleotide binding. Differences in amino acid sequences are localized near the amino terminus and in the carboxy half of the protein. Although the physiological functions of ARFs have not been precisely defined, their immunological localization to the Golgi is consistent with a role in the regulated orderly movement of newly synthesized proteins from the endoplasmic reticulum, through the Golgi system to their ultimate destination.     

Three-dimensional models of the GDP and GTP forms of the guanine nucleotide domain of Escherichia coli elongation factor Tu

Three-dimensional models of the GDP and GTP forms of the guanine nucleotide domain of Escherichia coli elongation factor Tu have been derived from the atomic coordinates of the trypsin-modified form of EF-Tu-GDP and by comparison with the ras p21 structures. The significance of the differences in the guanine nucleotide binding sites of EF-Tu and ras p21 are discussed. Crystallization of the EF-Tu-GMPPNP complex is reported.     

Protein synthesis elongation factors Tu and Tu.Ts from Caulobacter crescentus: sensitivity to kirromycin and activity in Q beta replicase.

The protein synthesis elongation factors Tu and Ts are responsible for binding aminoacyl-transfer ribonucleic acid (RNA) to the ribosome. In addition, they perform an undefined function, as the EF-Tu.Ts complex, in the RNA phage RNA replicases. In an effort to obtain insight into these two apparently unrelated roles, we purified the elongation factors from Caulobacter crescentus and compared them to the analogous Escherichia coli polypeptides. Although most physical and functional characteristics were found to be similar, significant differences were found in the molecular weight of EF-Ts and relative affinities of guanine nucleotides, sensitivity to trypsin cleavage, and rate of heat denaturation of EF-Tu. The antibiotic kirromycin was active with EF-Tu from both bacterial species. When C. crescentus EF-Tu.Ts was substituted for the E. coli elongation factors in Q beta phage RNA replicase, an enzyme capable of apparently normal RNA synthetic activity was formed.