Inhibitory mechanisms of antibiotics targeting elongation factor Tu.

Since the pioneering discovery of the inhibitory effects of kirromycin on bacterial elongation factor Tu (EF-Tu) more than 25 years ago [1], a great wealth of biological data has accumulated concerning protein biosynthesis inhibitors specific for EF-Tu. With the subsequent discovery of over two dozen naturally occurring EF-Tu inhibitors belonging to four different subclasses, EF-Tu has blossomed into an appealing antimicrobial target for rational drug discovery efforts. Very recently, independent crystal structure determinations of EF-Tu in complex with two potent antibiotics, aurodox and GE2270A, have provided structural explanations for the mode of action of these two compounds, and have set the foundation for the design of inhibitors with higher bioavailability, broader spectra, and greater efficacy.

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.

GE2270A-resistant mutations in elongation factor Tu allow productive aminoacyl-tRNA binding to EF-Tu.GTP.GE2270A complexes.

The antibiotic GE2270A prevents stable complex formation between elongation factor Tu (EF-Tu) and aminoacyl-tRNA (aatRNA). In Escherichia coli we characterized two mutant EF-Tu species with either G257S or G275A that lead to high GE2270A resistance in poly(Phe) synthesis, which at least partially explains the high resistance of EF-Tu1 from GE2270A producer Planobispora rosea to its own antibiotic. Both E. coli mutants were unexpectedly found to bind GE2270A nearly as well as wild-type (wt) EF-Tu in their GTP-bound conformations. Both G257S and G275A are in or near the binding site for the 3' end of aatRNA. The G257S mutation causes a 2.5-fold increase in affinity for aatRNA, whereas G275A causes a 40-fold decrease. In the presence of GE2270A, wt EF-Tu shows a drop in aatRNA affinity of at least four orders of magnitude. EF-Tu[G275S] and EF-Tu[G275A] curtail this drop to about two or one order, respectively. It thus appears that the resistance mutations do not prevent GE2270A from binding to EF-Tu.GTP and that the mutant EF-Tus may accommodate GE2270A and aatRNA simultaneously. Interestingly, in their GDP-bound conformations the mutant EF-Tus have much less affinity for GE2270A than wt EF-Tu. The latter is explained by a recent crystal structure of the EF-Tu.GDP.GE2270A complex, which predicts direct steric problems between GE2270A and the mutated G257S or G275A. These mutations may cause a dislocation of GE2270A in complex with GTP-bound EF-Tu, which then no longer prevents aatRNA binding as in the wt situation. Altogether, the data lead to the following novel resistance scenario. Upon arrival of the mutant EF-Tu.GTP.GE2270.aatRNA complex at the ribosomal A-site, the GTPase centre is triggered. The affinities of aatRNA and GE2270A for the GDP-bound EF-Tu are negligible; the former stays at the A-site for subsequent interaction with the peptidyltransferase centre and the latter two dissociate from the ribosome.

Structure and expression of elongation factor Tu from Bacillus stearothermophilus.

The tuf gene coding for elongation factor Tu (EF-Tu) of Bacillus stearothermophilus was cloned and sequenced. This gene maps in the same context as the tufA gene of Escherichia coli str operon. Northern-blot analysis and primer extension experiments revealed that the transcription of the tuf gene is driven from two promoter regions. One of these is responsible for producing a 4.9-kb transcript containing all the genes of B. stearothermophilus str operon and the other, identified adjacent to the stop codon of the fus gene and designated tufp, for producing a 1.3-kb transcript of the tuf gene only. In contrast to the situation in E. coli, the ratio between the transcription products was found to be about 10:1 in favour of the tuf gene transcript. This high transcription activity from the tufp promoter might be accounted for by the presence of an extremely A+T-rich block consisting of 29 nucleotides which immediately precedes the consensus -35 region of the promoter. A very similar tuf gene transcription strategy and the same tufp promoter organization with the identical A/T block were found in Bacillus subtilis. The tuf gene specifies a protein of 395 amino acid residues with a molecular mass of 43,290 Da, including the N-terminal methionine. A computer-generated three-dimensional homology model shows that all the structural elements essential for binding guanine nucleotides and aminoacyl-tRNA are conserved. The presence of serine at position 376 and a low affinity for kirromycin determined by zone-interference gel electrophoresis (Kd approximately 8 microM) and by polyacrylamide gel electrophoresis under non-denaturing conditions are in agreement with the reported resistance of this EF-Tu to the antibiotic. The replacement of the highly conserved Leu211 by Met was identified as a possible cause of pulvomycin resistance.

A growth-defective kirromycin-resistant EF-Tu Escherichia coli mutant and a spontaneously evolved suppression of the defect.

This study has investigated the cause of a growth-defect phenotype of a mutation in the elongation factor EF-Tu from Escherichia coli. An M13-based genetic retrieval system reported by Zeef and Bosch [Mol. Gen. Genet. 238 (1993) 252-260] was used to segregate and identify an extremely growth-defective kirromycin-resistant (KrR) tufA mutation, encoding Gln124-->Lys (Q124K), from a KrR parent strain. This original strain also contained mutations, 124com1 and 124com2, that appear to have evolved to suppress the Q124K tufA mutation. In this communication we present these M13-based genetic experiments together with additional genetic and protein characterization experiments to clarify the basis of this complementation. The data indicate that the serious growth defect of Q124K originates from a defective GTP/GDP interaction. The GTP/GDP binding and GTP hydrolysis characteristics of ET-Tu Q124K were different from wild-type EF-Tu and especially of another KrR EF-Tu mutant A375T. In line with this, 124com1 specifically complemented EF-Tu Q124K, whereas the growth defects of strains containing EF-Tu mutated at aa 375 were aggravated. We also show that strains containing the segregated tufA Q124K mutation formed filaments.

Antibiotic resistance mechanisms of mutant EF-Tu species in Escherichia coli.

Analysis of antibiotic-resistant EF-Tu mutants has revealed a connection between resistance and structural elements that participate in the GTPase switching mechanism. Both random and site-directed mutagenesis methods have yielded sets of purified mutant EF-Tu resistant to kirromycin (kirT) or pulvomycin (pulT). All kirT mutations cluster in the interface of domain 1 and 3 of EF-Tu in its GTP-bound conformation, not in that of EF-Tu.GDP. Other evidence also suggests that kirromycin binds to the interface of wild-type EF-Tu, thereby jamming the GTPase switch. Various functional studies reveal two subsequent resistance mechanisms. The first hinders kirromycin binding to EF-Tu.GTP and the second occurs after GTP hydrolysis by rejection of bound kirromycin. All pulT mutations cluster in the three-domain junction interface of EF-Tu. GTP (which is an open hole in EF-Tu.GDP) and destabilize a salt-bridge network. Pulvomycin may bind nearby and overlap with tRNA binding. Mutations show that a D99-R230 salt bridge is not essential for the transduction of the GTPase switch signal from domain 1. In vivo and in vitro studies reveal that pulvomycin sensitivity is dominant over resistance. This demands a revision of the current view of the mechanism of pulvomycin inhibition of protein synthesis and may support a translation model with two EF-Tus on the ribosome. Several mutant EF-Tu species display altered behaviour towards aminoacyl-tRNA with interesting effects on translational accuracy. KirT EF-Tu(A375T) is able to reverse the streptomycin-dependent phenotype of a ribosomal protein S12 mutant strain to streptomycin sensitivity.

Phosphorylation of elongation factor Tu prevents ternary complex formation.

The elongation factor Tu (EF-Tu) is a member of the GTP/GDP-binding proteins and interacts with various partners during the elongation cycle of protein biosynthesis thereby mediating the correct binding of amino-acylated transfer RNA (aa-tRNA) to the acceptor site (A-site) of the ribosome. After GTP hydrolysis EF-Tu is released in its GDP-bound state. In vivo, EF-Tu is post-translationally modified by phosphorylation. Here we report that the phosphorylation of EF-Tu by a ribosome associated kinase activity is drastically enhanced by EF-Ts. The antibiotic kirromycin, known to block EF-Tu function, inhibits the modification. This effect is specific, since kirromycin-resistant mutants do become phosphorylated in the presence of the antibiotic. On the other hand, phosphorylated wild-type EF-Tu does not bind kirromycin. Most interestingly, the phosphorylation of EF-Tu abolishes its ability to bind aa-tRNA. In the GTP conformation the site of modification is located at the interface between domains 1 and 3 and is involved in a strong interdomain hydrogen bond. Introduction of a charged phosphate group at this position will change the interaction between the domains, leading to an opening of the molecule reminiscent of the GDP conformation. A model for the function of EF-Tu phosphorylation in protein biosynthesis is presented.

Synergism between the GTPase activities of EF-Tu.GTP and EF-G.GTP on empty ribosomes. Elongation factors as stimulators of the ribosomal oscillation between two conformations.

A remarkable positive cooperativity between the GTPase activities of EF-Tu and EF-G on empty ribosomes from Escherichia coli has been discovered. This cooperativity implies a decrease of the corresponding apparent KM values of the empty ribosome for either elongation factor: from more than 10 microM to 0.5 microM for EF-Tu.GTP by the addition of 0.25 microM EF-G and from 0.7 microM to 0.5 microM for EF-G.GTP by the addition of 3 microM EF-Tu. In a further analysis of this phenomenon, the effects of various specific antibiotics were studied: thiostrepton, fusidic acid, tetracycline, pulvomycin and kirromycin appeared to inhibit the synergistic effect, whereas streptomycin was found to stimulate it. Even in the present minimal system the ribosomes respond to the above-mentioned antibiotics in a way surprisingly similar to that in the coupled system with mRNA and tRNAs. The cooperativity seems not to be due to a simultaneous binding of the two elongation factors to the ribosome as revealed by studying the effects of fusidic acid and kirromycin, and by band-shift experiments by means of gel electrophoresis under non-denaturing conditions. Our experimental data and the kinetic analysis of alternative models provide evidence that EF-Tu.GTP and EF-G.GTP interact sequentially with empty ribosomes that oscillate between two different conformations, one for each elongation factor. Apparently, ribosomes have an intrinsic property for oscillation as normally observed during protein synthesis with a frequency paced by the events of tRNA binding and translocation.

The structural and functional basis for the kirromycin resistance of mutant EF-Tu species in Escherichia coli.

A structural and functional understanding of resistance to the antibiotic kirromycin in Escherichia coli has been sought in order to shed new light on the functioning of the bacterial elongation factor Tu (EF-Tu), in particular its ability to act as a molecular switch. The mutant EF-Tu species G316D, A375T, A375V and Q124K, isolated by M13mp phage-mediated targeted mutagenesis, were studied. In this order the mutant EF-Tu species showed increasing resistance to the antibiotic as measured by poly(U)-directed poly(Phe) synthesis and intrinsic GTPase activities. The K'd values for kirromycin binding to mutant EF-Tu.GTP and EF-Tu.GDP increased in the same order. All mutation sites cluster in the interface of domains 1 and 3 of EF-Tu.GTP, not in that of EF-Tu.GDP. Evidence is presented that kirromycin binds to this interface of wild-type EF-Tu.GTP, thereby jamming the conformational switch of EF-Tu upon GTP hydrolysis. We conclude that the mutations result in two separate mechanisms of resistance to kirromycin. The first inhibits access of the antibiotic to its binding site on EF-Tu.GTP. A second mechanism exists on the ribosome, when mutant EF-Tu species release kirromycin and polypeptide chain elongation continues.

Divergent effects of fluoroaluminates on the peptide chain elongation factors EF-Tu and EF-G as members of the GTPase superfamily.

Fluoraluminates are thought to mimic the gamma-phosphate of GTP and thus, together with GDP, perturb the functioning of heterotrimeric GTP-binding G-proteins. Here we show they do inhibit the ribosome-stimulated GTPase activity of EF-G from Escherichia coli via the formation of a stable complex with EF-G-GDP and ribosomes. In contrast, no perturbed interactions were observed in a similar ribosomal complex with EF-Tu. Interestingly, in the absence of ribosomes both EF-Tu an EF-G remain totally unaffected by fluoraluminates. For members of the GTPase superfamily such differential effects have not been described before.

Elongation factors in protein synthesis.

Recent discoveries of elongation factor-related proteins have considerably complicated the simple textbook scheme of the peptide chain elongation cycle. During growth and differentiation the cycle may be regulated not only by factor modification but also factor replacement. In addition, rare tRNAs may have their own rare factor proteins. A special case is the acquisition of resistance by bacteria to elongation factor-directed antibiotics. Pertinent data from the literature and our own work with Escherichia coli and Streptomyces are discussed. The GTP-binding domain of EF-Tu has been studied extensively, but little molecular detail is available on the interactions with its other ligands or effectors, or on the way they are affected by the GTPase switch signal. A growing number of EF-Tu mutants obtained by ourselves and others are helping us in testing current ideas. We have found a synergistic effect between EF-Tu and EF-G in their uncoupled GTPase reactions on empty ribosomes. Only the EF-G reaction is perturbed by fluoroaluminates.

Fluoroaluminates do not affect the guanine-nucleotide binding centre of the peptide chain elongation factor EF-Tu.

EF-Tu is often referred to as a model for guanine-nucleotide-binding regulatory proteins (G-proteins), since X-ray diffraction analysis of its GTP-binding domain shows a detailed location of the 'consensus' amino acid sequences involved in nucleotide binding. Fluoroaluminates are thought to mimick the gamma-phosphate in the GTPase centre on account of their activating effect on a variety of GTP binding proteins. In the case of EF-Tu, we could find no such effects on the basis of at least three independent functional assays. We did notice, however, complicating interactions between free nucleotides, fluoroaluminates and other ligands. By consequence, if indeed AlF4- behaves as a gamma-phosphate analogue in G-proteins, then EF-Tu must have a different GDP/GTP binding site, despite of the conserved consensus sequences.

Experimental simulation of oxygen profiles and their influence on baker's yeast production: I. One-fermentor system.

In production-scale bioreactors microorganisms are exposed to a continually changing environment. This may cause loss of viability, reduction of the yield of biomass or desired metabolites, and an increase in the formation of by-products. In fed-batch production of baker's yeast, profiles may occur in substrate and oxygen concentrations and in pH. This article deals with the influence of a periodically changing oxygen concentration on the growth of baker's yeast in a continuous culture. Also, influences on the production of ethanol, glycerol, acetic acid, and on the composition of the cells were investigated. It was found that relatively fast fluctuations between oxygen-unlimited and oxygen-limited conditions with a frequency of 1 or 2 min had a distinct influence on the biomass and metabolite production. However, RNA, protein, and carbohydrate contents measured in cells exposed to fluctuations differed little from those in cells from an oxygen-unlimited or an oxygen-limited culture. The respiration and fermentation capacities of cells exposed to fluctuations can be larger than the capacities of cells grown under oxygen-unlimited conditions.