Engineered EF-Tu and tRNA-Based FRET Screening Assay to Find Inhibitors of Protein Synthesis in Bacteria.

Antibiotic-resistant infections that do not respond to available drugs are becoming more common. Methicillin-resistant Staphylococcus aureus, carbapenem-resistant enterobacteria ("superbugs"), and many others pose a continuous threat to public health. To provide tools to combat such deadly infections, we present in this study a homogeneous assay focused on an insufficiently addressed molecular interaction linked to ribosomal translation. We show that a fluorescence resonance energy transfer (FRET) based screening assay can identify antibiotic molecules that inhibit ternary complex (EF-Tu:tRNA:GTP complex) formation, and therefore, protein synthesis in bacteria. Specifically engineered Escherichia coli EF-Tu and tRNAPhe are used to prepare two key components of this assay: (1) Cy5-EF-Tu:GTP and (2) Cy3-Phe-tRNAPhe. When mixed and Cy3 is excited at 532 nm, increased Cy5 fluorescence intensity is observed at 665 nm due to ternary complex formation and FRET. If the same assay is carried out in presence of an inhibitor, such as GE2270A (a known inhibitor of the EF-Tu-tRNA interaction), fluorescence intensity is significantly diminished. To establish proof of principle and to show the adaptability of this assay to high throughput screening (HTS), we analyzed the effect of different classes of antibiotics, including beta-lactams, quinolone compounds, and protein synthesis inhibitors, on fluorescence. The assay was done in a 96-well microplate. We observed inhibition by GE2270A, and no effect of nineteen other tested antibiotics, confirming the ability of this FRET assay to serve as a screen for potential inhibitor molecules of ternary complex formation from libraries of compounds.

[Proteomic analysis of contaminants in recombinant membrane hemeproteins expressed in E. coli and isolated by metal affinity chromatography].

Contaminating proteins have been identified by "shotgun" proteomic analysis in 14 recombinant preparations of human membrane heme- and flavoproteins expressed in Escherichia coli and purified by immobilized metal ion affinity chromatography. Immobilized metal ion affinity chromatography of ten proteins was performed on Ni2+-NTA-sepharose 6B, and the remaining four proteins were purified by ligand affinity chromatography on 2',5'-ADP-sepharose 4B. Proteomic analysis allowed to detect 50 protein impurities from E. coli. The most common contaminant was Elongation factor Tu2. It is characterized by a large dipole moment and a cluster arrangement of acidic amino acid residues that mediate the specific interaction with the sorbent. Peptidyl prolyl-cis-trans isomerase SlyD, glutamine-fructose-6-phosphate aminotransferase, and catalase HPII that contained repeating HxH, QxQ, and RxR fragments capable of specific interaction with the sorbent were identified among the protein contaminants as well. GroL/GroS chaperonins were probably copurified due to the formation of complexes with the target proteins. The Ni2+ cations leakage from the sorbent during lead to formation of free carboxyl groups that is the reason of cation exchanger properties of the sorbent. This was the putative reason for the copurification of basic proteins, such as the ribosomal proteins of E. coli and the widely occurring uncharacterized protein YqjD. The results of the analysis revealed variation in the contaminant composition related to the type of protein expressed. This is probably related to the reaction of E. coli cell proteome to the expression of a foreign protein. We concluded that the nature of the protein contaminants in a preparation of a recombinant protein purified by immobilized metal ion affinity chromatography on a certain sorbent could be predicted if information on the host cell proteome were available.

Cloning and characterization of EF-Tu and EF-Ts from Pseudomonas aeruginosa.

We have cloned genes encoding elongation factors EF-Tu and EF-Ts from Pseudomonas aeruginosa and expressed and purified the proteins to greater than 95% homogeneity. Sequence analysis indicated that P. aeruginosa EF-Tu and EF-Ts are 84% and 55% identical to E. coli counterparts, respectively. P. aeruginosa EF-Tu was active when assayed in GDP exchange assays. Kinetic parameters for the interaction of EF-Tu with GDP in the absence of EF-Ts were observed to be K M = 33 µM, k cat (obs) = 0.003 s(-1), and the specificity constant k cat (obs)/K M was 0.1 × 10(-3) s(-1) µM(-1). In the presence of EF-Ts, these values were shifted to K M = 2 µM, k cat (obs) = 0.005 s(-1), and the specificity constant k(cat)(obs)/K M was 2.5 × 10(-3) s(-1) µM(-1). The equilibrium dissociation constants governing the binding of EF-Tu to GDP (K GDP) were 30-75 nM and to GTP (K GTP) were 125-200 nM. EF-Ts stimulated the exchange of GDP by EF-Tu 10-fold. P. aeruginosa EF-Tu was active in forming a ternary complex with GTP and aminoacylated tRNA and was functional in poly(U)-dependent binding of Phe-tRNA(Phe) at the A-site of P. aeruginosa ribosomes. P. aeruginosa EF-Tu was active in poly(U)-programmed polyphenylalanine protein synthesis system composed of all P. aeruginosa components.

Identifying ligand-binding hot spots in proteins using brominated fragments.

High-quality crystals of Thermus thermophilus EF-Tu in the GTP-bound conformation at 1.7-2.7 Šresolution were used to test 18 small organic molecules, all brominated for confident identification in the anomalous difference maps. From this relatively small collection, it was possible to identify a small molecule bound in the functionally important tRNA CCA-end binding pocket. The antibiotic GE2270 A is known to interact with the same pocket in EF-Tu and to disrupt the association with tRNA. Bromide could be located from peaks in the anomalous map in data truncated to very low resolution without refining the structure. Considering the speed with which diffraction data can be collected today, it is proposed that it is worthwhile to collect the extra data from fragment screens while crystals are at hand to increase the knowledge of biological function and drug binding in an experimental structural context.

Comparative study of the life cycle dependent post-translation modifications of protein synthesis elongation factor Tu present in the membrane proteome of streptomycetes and mycobacteria.

We present the results of analysis of membrane phosphoproteomes from individual morphological stages of Streptomyces coelicolor that reflect developmentally dependent heterogeneity and phosphorylation of intrinsic and externally added purified Strepomyces aureofaciens EF-Tu. Fast growing nonpathogenic Mycobacterium smegmatis was used as a non-differentiating actinomycetes comparative model. Streptomycetes membrane fraction was found to contain protein kinase(s) catalyzing phosphorylation of both its own and an externally added EF-Tu, whereas Mycobacterium membrane fraction contains protein kinase phosphorylating only its own EF-Tu.

Properties of Escherichia coli EF-Tu mutants designed for fluorescence resonance energy transfer from tRNA molecules.

Here we describe the design, preparation and characterization of 10 EF-Tu mutants of potential utility for the study of Escherichia coli elongation factor Tu (EF-Tu) interaction with tRNA by a fluorescence resonance energy transfer assay. Each mutant contains a single cysteine residue at positions in EF-Tu that are proximal to tRNA sites within the aminoacyl-tRNA.EF-Tu.GTP ternary complex that have previously been labeled with fluorophores. These positions fall in the 323-326 and 344-348 regions of EF-Tu, and at the C terminus. The EF-Tus were isolated as N-terminal fusions to glutathione S-transferase (GST), which was cleaved to yield intact EF-Tus. The mutant EF-Tus were tested for binding to GDP, binding to tRNA in gel retardation and protection assays, and activity in poly-U translation in vitro. The results indicate that at least three EF-Tu mutants, K324C, G325C and E348C, are suitable for further studies. Remarkably, GST fusions that were not cleaved were also active in the various assays, despite the N-terminal fusion.

Functional metaproteome analysis of protein extracts from contaminated soil and groundwater.

Using proteins from soil or groundwater as functional biomarkers requires efficient extraction. We developed an extraction method in which the separation of proteins from the inorganic and organic constituents of the soil matrix was achieved by a combination of 0.1 M NaOH treatment and phenol extraction. Incubation with NaOH released humic acids and proteins from soil minerals, and simultaneously, disrupted microorganisms. The subsequent phenol extraction separated the proteins from the humic organic matter. Protein extracts were applied to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 2D-electrophoresis (2-DE). Spots and bands were excised and individual proteins identified by liquid chromatography online linked to mass spectrometry (MS) via electrospray ionization source (LC-ESI-MS). To assess the suitability of the method for the functional analysis of environmental metaproteomes, it was applied to soil that had been enriched in chlorophenoxy acid-degrading bacteria by incubation with 2,4-dichlorophenoxy acetic acid (2,4-D) for 22 days. The method was also used to analyze groundwater from the aquifer of a chlorobenzene-contaminated site. The identification of enzymes such as chlorocatechol dioxygenases was consistent with bacterial metabolic pathways expected to be expressed in these samples. The protocol enabled thus the analysis of the metaproteome of soil and groundwater samples. It thereby provides a means to study the diversity of environmental microbial communities while addressing functional aspects more directly than metagenome or even metatranscriptome analysis.

Chloroplast protein synthesis elongation factor, EF-Tu, reduces thermal aggregation of rubisco activase.

Chloroplast protein synthesis elongation factor, EF-Tu, has been implicated in heat tolerance in maize. The recombinant precursor of this protein, pre-EF-Tu, has been found to exhibit chaperone activity and protect heat-labile proteins, such as citrate synthase and malate dehydrogenase, from thermal aggregation. Chloroplast EF-Tu is highly conserved and it is possible that the chaperone activity of this protein is not species-specific. In this study, we investigated the effect of native wheat pre-EF-Tu on thermal aggregation of rubisco activase. Additionally, we investigated the effect of native and recombinant maize pre-EF-Tu on activase aggregation. Activase was chosen because it displays an exceptional sensitivity to thermal aggregation and constrains photosynthesis at high temperature. The native precursors of both wheat and maize EF-Tu displayed chaperone activity, as shown by the capacity of both proteins to reduce thermal aggregation of rubisco activase in vitro. Similarly, the recombinant maize pre-EF-Tu protected activase from thermal aggregation. This is the first report on chaperone activity of native pre-EF-Tu and the first evidence for thermal protection of a photosynthetic enzyme by this putative chaperone. The results are consistent with the hypothesis that chloroplast EF-Tu plays a functional role in heat tolerance by acting as a molecular chaperone.

Immune evasion of the human pathogen Pseudomonas aeruginosa: elongation factor Tuf is a factor H and plasminogen binding protein.

Pseudomonas aeruginosa is an opportunistic human pathogen that can cause a wide range of clinical symptoms and infections that are frequent in immunocompromised patients. In this study, we show that P. aeruginosa evades human complement attack by binding the human plasma regulators Factor H and Factor H-related protein-1 (FHR-1) to its surface. Factor H binds to intact bacteria via two sites that are located within short consensus repeat (SCR) domains 6-7 and 19-20, and FHR-1 binds within SCR domain 3-5. A P. aeruginosa Factor H binding protein was isolated using a Factor H affinity matrix, and was identified by mass spectrometry as the elongation factor Tuf. Factor H uses the same domains for binding to recombinant Tuf and to intact bacteria. Factor H bound to recombinant Tuf displayed cofactor activity for degradation of C3b. Similarly Factor H bound to intact P. aeruginosa showed complement regulatory activity and mediated C3b degradation. This acquired complement control was rather effective and acted in concert with endogenous proteases. Immunolocalization identified Tuf as a surface protein of P. aeruginosa. Tuf also bound plasminogen, and Tuf-bound plasminogen was converted by urokinase plasminogen activator to active plasmin. Thus, at the bacterial surface Tuf acts as a virulence factor and binds the human complement regulator Factor H and plasminogen. Acquisition of host effector proteins to the surface of the pathogen allows complement control and may facilitate tissue invasion.

Characterization of p43(ARF), a derivative of the p43 component of multiaminoacyl-tRNA synthetase complex released during apoptosis.

In human, nine aminoacyl tRNA synthetases are associated with the three auxiliary proteins, p18, p38, and p43, to form a stable multiprotein complex. The p43 component, which has a potent tRNA binding capacity, is associated to the complex via its N-terminal moiety. This protein is also the precursor of the endothelial monocyte-activating polypeptide II (p43(EMAPII), corresponding to the C-terminal moiety of p43), a cytokine generated during apoptosis. Here we examined the cellular pathway that, starting from the p43 subunit of the complex, leads to this extracellular cytokine. We identified a new intermediate in this pathway, named p43(ARF) for Apoptosis-released Factor. This intermediate is produced in cellulo by proteolytic cleavage of endogenous p43 and is rapidly recovered in the culture medium. This p43 derivative was purified from the medium of human U937 cells subjected to serum starvation. It contains 40 additional N-terminal amino acid residues as compared with the cytokine p43(EMAPII) and may be generated by a member of the matrix metalloproteinase family. Recombinant p43(ARF) is a monomer in solution and binds tRNA with a Kd of approximately 6 nM, 30-fold lower than that of p43. Highly purified p43(ARF) or p43(EMAPII) do not stimulate the expression of E-selectin by human umbilical vein endothelial cells. Our results suggest that the cleavage of p43 and its cellular delocalization, and thus the release of this tRNA binding subunit from the complex, is one of the molecular mechanisms leading to the shut down of protein synthesis in apoptosis.

Bacterial elongation factors EF-Tu, their mutants, chimeric forms, and domains: isolation and purification.

Prokaryotic elongation factors EF-Tu form a family of homologous, three-domain molecular switches catalyzing the binding of aminoacyl-tRNAs to ribosomes during the process of mRNA translation. They are GTP-binding proteins, or GTPases. Binding of GTP or GDP regulates their conformation and thus their activity. Because of their particular structure and regulation, various activities (also outside of the translation system) and a relative abundance they represent attractive tools for studies of many basic but still not fully understood mechanisms both of the translation process, the structure-function relationships in EF-Tu molecules themselves and proteins and energy transduction mechanisms in general. The review critically summarizes procedures for the isolation and purification of native and engineered eubacterial elongation factors EF-Tu and their mutants on a large as well as small scale. Current protocols for the purification of both native and polyHis-tagged or glutathione-S-transferase (GST)-tagged EF-Tu proteins and their variants using conventional procedures and the Ni-NTA-Agarose or Glutathione Sepharose are presented.

Protein synthesis elongation factor Tu present in spores of Streptomyces coelicolor can be phosphorylated in vitro by the spore protein kinase.

In vitro phosphorylation of EF-Tu was shown in cell-free extract from dormant spores of Streptomyces coelicolor by a protein kinase present in spores. EF-Tu phosphorylation was observed on both intrinsic S. coelicolor factor and externally added purified EF-Tu from S. aureofaciens, on two isoforms. Putative serine and threonine residues as potential phosphorylation targets were determined in primary sequence and demonstrated on 3D structure model of EF-Tu.

Post-translational modification(s) and cell distribution of Streptomyces aureofaciens translation elongation factor Tu overproduced in Escherichia coli.

We cloned EF-Tu from Streptomyces aureofaciens on a pET plasmid and overproduced it using the T7 RNA polymerase system in Escherichia coli. Streptomyces EF-Tu represented more than 40% of the total cell protein and was stored mostly in inclusion bodies formed apically at both ends of E. coli cells. Analysis of the inclusion bodies by transmission and scanning electron microscopy did not reveal any internal or surface ultrastructures. We developed the method for purification of S. aureofaciens EF-Tu from isolated inclusion bodies based on the ability of the protein to aggregate spontaneously. EF-Tu present in inclusion bodies was not active in GDP binding. Purified protein showed a similar charge heterogeneity as EF-Tu isolated from the mycelium of S. aureofaciens and all of the isoforms reacted with EF-Tu antibodies. All isoforms also reacted with monoclonal antibodies against O-phosphoserine and O-phosphothreonine.

The N terminus of bacterial elongation factor Tu elicits innate immunity in Arabidopsis plants.

Innate immunity is based on the recognition of pathogen-associated molecular patterns (PAMPs). Here, we show that elongation factor Tu (EF-Tu), the most abundant bacterial protein, acts as a PAMP in Arabidopsis thaliana and other Brassicaceae. EF-Tu is highly conserved in all bacteria and is known to be N-acetylated in Escherichia coli. Arabidopsis plants specifically recognize the N terminus of the protein, and an N-acetylated peptide comprising the first 18 amino acids, termed elf18, is fully active as inducer of defense responses. The shorter peptide, elf12, comprising the acetyl group and the first 12 N-terminal amino acids, is inactive as elicitor but acts as a specific antagonist for EF-Tu-related elicitors. In leaves of Arabidopsis plants, elf18 induces an oxidative burst and biosynthesis of ethylene, and it triggers resistance to subsequent infection with pathogenic bacteria.

Comparative proteome analysis of Mycobacterium tuberculosis grown under aerobic and anaerobic conditions.

Data are presented from two-dimensional (2-D) PAGE analysis of Mycobacterium tuberculosis strain Harlingen grown during aerobic and anaerobic culture, according to a modified Wayne dormancy model. M. tuberculosis cultures were grown to the transition point between exponential growth and stationary phase in the presence of oxygen (7 days) and then part of the cultures was shifted to anaerobic conditions for 16 days. Growth declined similarly during aerobic and anaerobic conditions, whereas the ATP consumption rapidly decreased in the anaerobic cultures. 2-D PAGE revealed 50 protein spots that were either unique to, or more abundant during, anaerobic conditions and 16 of these were identified by MALDI-TOF. These proteins were the alpha-crystalline homologue (HspX), elongation factor Tu (Tuf), GroEL2, succinyl-CoA : 3-oxoacid-CoA transferase (ScoB), mycolic acid synthase (CmaA2), thioredoxin (TrxB2), beta-ketoacyl-ACP synthase (KasB), l-alanine dehydrogenase (Ald), Rv2005c, Rv2629, Rv0560c, Rv2185c and Rv3866. Some protein spots were found to be proteolytic fragments, e.g. HspX and GroEL2. These data suggest that M. tuberculosis induces expression of about 1 % of its genes in response to dormancy.

Immobilization of a novel antibacterial agent on solid phase and subsequent isolation of EF-Tu.

Screening of our compound collection identified PNU-92560, a 2-[1,3,4]thiadiazolo[3,2-a]pyrimidine-6-carboxamide, as a novel antibacterial agent. Extensive analogue development identified that the 2-position of the thiadiazole could be functionalized with a linker that would allow the compound to be attached to a solid support. The extreme insolubility of the analogues prevented the mechanism of action for these compounds to be determined utilizing traditional methodology. The solid-supported compounds were utilized as affinity columns to identify elongation factor Tu (EF-Tu) as a putative target for this class of compounds. The activity of the compounds in a metabolic labeling experiments and in translation assay supports the identity of the target for these compounds to be EF-Tu.

Isolation of Qbeta polymerase complexes containing mutant species of elongation factor Tu.

The RNA genome of coliphage Qbeta is replicated by a complex of four proteins, one of them being the translation elongation factor Tu. The role of EF-Tu in this RNA polymerase complex is still unclear, but the obligate presence of translationally functional EF-Tu in the cell hampers the use of conventional mutational analysis. Therefore, we designed a system based on affinity chromatography and could separate two types of complexes by placing an affinity tag on mutated EF-Tu species. Thus, we were able to show a direct link between the vital tRNA binding property of EF-Tu and polymerase activity.

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.

Isolation of chimaeric forms of elongation factor EF-Tu by affinity chromatography.

Six different recombinant chimaeric forms of a three-domain protein, proteosynthetic elongation factor Tu (EF-Tu), composed of domains of EF-Tu of mesophilic (Escherichia coli) and thermophilic (Bacillus stearothermophilus) origin as well as free N-terminal domains of EF-Tu, and the whole recombinant EF-Tus of both organisms were prepared and isolated by the GST (glutathione S-transferase) fusion technology. Several modifications in the standard isolation and purification procedures are described that proved necessary to obtain the proteins in a purified and undegraded form.

Efficient separation of Thermus aquaticus EF-Tu functional complexes.

A new method for fast separation of the main functional complexes of the elongation factor Tu from Thermus aquaticus has been developed. Binary complexes EF-Tu * GDP and EF-Tu * GDPNP as well as the ternary complex EF-Tu * GDPNP * Leu approximately tRNA were separated from each other by means of HPLC on a hydrophobic sorbent TSK-Gel Phenyl 5PW in a reverse gradient of ammonium sulfate. This technique is suitable for monitoring EF-Tu activity, characterisation of the ratio between different EF-Tu forms in cell extracts, and isolation of individual EF-Tu complexes for structural and functional investigations. In order to illustrate the potentials of the method, we used HPLC on a TSK-Gel Phenyl 5PW matrix to determine the ratio between affinities of GDP and GDPNP for EF-Tu. We found that K(a)(GDP) is about 27 times higher than K(a)(GDPNP) at 37 degrees C, the value being close to the one reported for Thermus thermophilus EF-Tu.