Structures of the Staphylococcus aureus ribosome inhibited by fusidic acid and fusidic acid cyclopentane.

The antibiotic fusidic acid (FA) is used to treat Staphylococcus aureus infections. It inhibits protein synthesis by binding to elongation factor G (EF-G) and preventing its release from the ribosome after translocation. While FA, due to permeability issues, is only effective against gram-positive bacteria, the available structures of FA-inhibited complexes are from gram-negative model organisms. To fill this knowledge gap, we solved cryo-EM structures of the S. aureus ribosome in complex with mRNA, tRNA, EF-G and FA to 2.5 Å resolution and the corresponding complex structures with the recently developed FA derivative FA-cyclopentane (FA-CP) to 2.0 Å resolution. With both FA variants, the majority of the ribosomal particles are observed in chimeric state and only a minor population in post-translocational state. As expected, FA binds in a pocket between domains I, II and III of EF-G and the sarcin-ricin loop of 23S rRNA. FA-CP binds in an identical position, but its cyclopentane moiety provides additional contacts to EF-G and 23S rRNA, suggesting that its improved resistance profile towards mutations in EF-G is due to higher-affinity binding. These high-resolution structures reveal new details about the S. aureus ribosome, including confirmation of many rRNA modifications, and provide an optimal starting point for future structure-based drug discovery on an important clinical drug target.

Polydatin and Nicotinamide Rescue the Cellular Phenotype of Mitochondrial Diseases by Mitochondrial Unfolded Protein Response (mtUPR) Activation.

Primary mitochondrial diseases result from mutations in nuclear DNA (nDNA) or mitochondrial DNA (mtDNA) genes, encoding proteins crucial for mitochondrial structure or function. Given that few disease-specific therapies are available for mitochondrial diseases, novel treatments to reverse mitochondrial dysfunction are necessary. In this work, we explored new therapeutic options in mitochondrial diseases using fibroblasts and induced neurons derived from patients with mutations in the GFM1 gene. This gene encodes the essential mitochondrial translation elongation factor G1 involved in mitochondrial protein synthesis. Due to the severe mitochondrial defect, mutant GFM1 fibroblasts cannot survive in galactose medium, making them an ideal screening model to test the effectiveness of pharmacological compounds. We found that the combination of polydatin and nicotinamide enabled the survival of mutant GFM1 fibroblasts in stress medium. We also demonstrated that polydatin and nicotinamide upregulated the mitochondrial Unfolded Protein Response (mtUPR), especially the SIRT3 pathway. Activation of mtUPR partially restored mitochondrial protein synthesis and expression, as well as improved cellular bioenergetics. Furthermore, we confirmed the positive effect of the treatment in GFM1 mutant induced neurons obtained by direct reprogramming from patient fibroblasts. Overall, we provide compelling evidence that mtUPR activation is a promising therapeutic strategy for GFM1 mutations.

The Structural and Molecular Mechanisms of Mycobacterium tuberculosis Translational Elongation Factor Proteins.

Targeting translation factor proteins holds promise for developing innovative anti-tuberculosis drugs. During protein translation, many factors cause ribosomes to stall at messenger RNA (mRNA). To maintain protein homeostasis, bacteria have evolved various ribosome rescue mechanisms, including the predominant trans-translation process, to release stalled ribosomes and remove aberrant mRNAs. The rescue systems require the participation of translation elongation factor proteins (EFs) and are essential for bacterial physiology and reproduction. However, they disappear during eukaryotic evolution, which makes the essential proteins and translation elongation factors promising antimicrobial drug targets. Here, we review the structural and molecular mechanisms of the translation elongation factors EF-Tu, EF-Ts, and EF-G, which play essential roles in the normal translation and ribosome rescue mechanisms of Mycobacterium tuberculosis (Mtb). We also briefly describe the structure-based, computer-assisted study of anti-tuberculosis drugs.

Multi-Channel smFRET study reveals a Compact conformation of EF-G on the Ribosome.

While elongation factor G (EF-G) is crucial for ribosome translocation, the role of its GTP hydrolysis remains ambiguous. EF-G's indispensability is further exemplified by the phosphorylation of human eukaryotic elongation factor 2 (eEF2) at Thr56, which inhibits protein synthesis globally, but its exact mechanism is not clear. In this study, we developed a multi-channel single-molecule FRET (smFRET) microscopy methodology to examine the conformational changes of E. coli EF-G induced by mutations that closely aligned with eEF2's Thr56 residue. We utilized Alexa 488/594 double-labeled EF-G to catalyze the translocation of fMet-Phe-tRNAPhe-Cy3 inside Cy5-L27 labeled ribosomes, allowing us to probe both processes within the same complex. Our findings indicate that in the presence of either GTP or GDPCP, wild-type EF-G undergoes a conformational extension upon binding to the ribosome to promote normal translocation. On the other hand, T48E and T48V mutations did not affect GTP/GDP binding or GTP hydrolysis, but impeded Poly(Phe) synthesis and caused EF-G to adopt a unique compact conformation, which wasn't observed when the mutants interact solely with the sarcin/ricin loop. This study provides new insights into EF-G's adaptability and sheds light on the modification mechanism of human eEF2.

Unlocking translational machinery for antitubercular drug development.

Targeting translational factor proteins (TFPs) presents significant promise for the development of innovative antitubercular drugs. Previous insights from antibiotic binding mechanisms and recently solved 3D crystal structures of Mycobacterium tuberculosis (Mtb) elongation factor thermo unstable-GDP (EF-Tu-GDP), elongation factor thermo stable-EF-Tu (EF-Ts-EF-Tu), and elongation factor G-GDP (EF-G-GDP) have opened up new avenues for the design and development of potent antituberculosis (anti-TB) therapies.

Mechanistic dissection of premature translation termination induced by acidic residues-enriched nascent peptide.

Ribosomes polymerize nascent peptides through repeated inter-subunit rearrangements between the classic and hybrid states. The peptidyl-tRNA, the intermediate species during translation elongation, stabilizes the translating ribosome to ensure robust continuity of elongation. However, the translation of acidic residue-rich sequences destabilizes the ribosome, leading to a stochastic premature translation cessation termed intrinsic ribosome destabilization (IRD), which is still ill-defined. Here, we dissect the molecular mechanisms underlying IRD in Escherichia coli. Reconstitution of the IRD event reveals that (1) the prolonged ribosome stalling enhances IRD-mediated translation discontinuation, (2) IRD depends on temperature, (3) the destabilized 70S ribosome complex is not necessarily split, and (4) the destabilized ribosome is subjected to peptidyl-tRNA hydrolase-mediated hydrolysis of the peptidyl-tRNA without subunit splitting or recycling factors-mediated subunit splitting. Collectively, our data indicate that the translation of acidic-rich sequences alters the conformation of the 70S ribosome to an aberrant state that allows the noncanonical premature termination.

Comparing FRET Pairs that Report on Intersubunit Rotation in Bacterial Ribosomes.

Mediated by elongation factor G (EF-G), ribosome translocation along mRNA is accompanied by rotational movement between ribosomal subunits. Here, we reassess whether the intersubunit rotation requires GTP hydrolysis by EF-G or can occur spontaneously. To that end, we employ two independent FRET assays, which are based on labeling either ribosomal proteins (bS6 and bL9) or rRNAs (h44 of 16S and H101 of 23S rRNA). Both FRET pairs reveal three FRET states, corresponding to the non-rotated, rotated and semi-rotated conformations of the ribosome. Both FRET assays show that in the absence of EF-G, pre-translocation ribosomes containing deacylated P-site tRNA undergo spontaneous intersubunit rotations between non-rotated and rotated conformations. While the two FRET pairs exhibit largely similar behavior, they substantially differ in the fraction of ribosomes showing spontaneous fluctuations. Nevertheless, instead of being an invariable intrinsic property of each FRET pair, the fraction of spontaneously fluctuating molecules changes in both FRET assays depending on experimental conditions. Our results underscore importance of using multiple FRET pairs in studies of ribosome dynamics and highlight the role of thermally-driven large-scale ribosome rearrangements in translation.

Decoding and Recoding of mRNA Sequences by the Ribosome.

Faithful translation of messenger RNA (mRNA) into protein is essential to maintain protein homeostasis in the cell. Spontaneous translation errors are very rare due to stringent selection of cognate aminoacyl transfer RNAs (tRNAs) and the tight control of the mRNA reading frame by the ribosome. Recoding events, such as stop codon readthrough, frameshifting, and translational bypassing, reprogram the ribosome to make intentional mistakes and produce alternative proteins from the same mRNA. The hallmark of recoding is the change of ribosome dynamics. The signals for recoding are built into the mRNA, but their reading depends on the genetic makeup of the cell, resulting in cell-specific changes in expression programs. In this review, I discuss the mechanisms of canonical decoding and tRNA-mRNA translocation; describe alternative pathways leading to recoding; and identify the links among mRNA signals, ribosome dynamics, and recoding.

Interplay between Inter-Subunit Rotation of the Ribosome and Binding of Translational GTPases.

Translational G proteins, whose release from the ribosome is triggered by GTP hydrolysis, regulate protein synthesis. Concomitantly with binding and dissociation of protein factors, translation is accompanied by forward and reverse rotation between ribosomal subunits. Using single-molecule measurements, we explore the ways in which the binding of translational GTPases affects inter-subunit rotation of the ribosome. We demonstrate that the highly conserved translation factor LepA, whose function remains debated, shifts the equilibrium toward the non-rotated conformation of the ribosome. By contrast, the catalyst of ribosome translocation, elongation factor G (EF-G), favors the rotated conformation of the ribosome. Nevertheless, the presence of P-site peptidyl-tRNA and antibiotics, which stabilize the non-rotated conformation of the ribosome, only moderately reduces EF-G binding. These results support the model suggesting that EF-G interacts with both the non-rotated and rotated conformations of the ribosome during mRNA translocation. Our results provide new insights into the molecular mechanisms of LepA and EF-G action and underscore the role of ribosome structural dynamics in translation.

3-Amino-Substituted Analogues of Fusidic Acid as Membrane-Active Antibacterial Compounds.

Fusidic acid (FA) is an antibiotic with high activity against Staphylococcus aureus; it has been used in clinical practice since the 1960s. However, the narrow antimicrobial spectrum of FA limits its application in the treatment of bacterial infections. In this regard, this work aims both at the study of the antimicrobial effect of a number of FA amines and at the identification of their potential biological targets. In this way, FA analogues containing aliphatic and aromatic amino groups and biogenic polyamine, spermine and spermidine, moieties at the C-3 atom, were synthesized (20 examples). Pyrazinecarboxamide-substituted analogues exhibit a high antibacterial activity against S. aureus (MRSA) with MIC ≤ 0.25 µg/mL. Spermine and spermidine derivatives, along with activity against S. aureus, also inhibit the growth and reproduction of Gram-negative bacteria Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa, and have a high fungicidal effect against Candida albicans and Cryptococcus neoformans. The study of the membrane activity demonstrated that the spermidine- and spermine-containing compounds are able to immerse into membranes and disorder the lipidsleading to a detergent effect. Moreover, spermine-based compounds are also able to form ion-permeable pores in the lipid bilayers mimicking the bacterial membranes. Using molecular docking, inhibition of the protein synthesis elongation factor EF-G was proposed, and polyamine substituents were shown to make the greatest contribution to the stability of the complexes of fusidic acid derivatives with biological targets. This suggests that the antibacterial effect of the obtained compounds may be associated with both membrane activity and inhibition of the elongation factor EF-G.

Drop-off-reinitiation at the amino termini of nascent peptides and its regulation by IF3, EF-G, and RRF.

In translation initiation in prokaryotes, IF3 recognizes the interaction between the initiator codon of mRNA and the anticodon of fMet-tRNAini and then relocates the fMet-tRNAini to an active position. Here, we have surveyed 328 codon-anticodon combinations for the preference of IF3. At the first and second base of the codon, only Watson-Crick base pairs are tolerated. At the third base, stronger base pairs, for example, Watson-Crick, are more preferred, but other types of base pairs, for example, G/U wobble, are also tolerated; weaker base pairs are excluded by IF3. When the codon-anticodon combinations are unfavorable for IF3 or the concentration of IF3 is too low to recognize any codon-anticodon combinations, IF3 fails to set the P-site fMet-tRNAini at the active position and causes its drop-off from the ribosome. Thereby, translation reinitiation occurs from the second aminoacyl-tRNA at the A site to yield a truncated peptide lacking the amino-terminal fMet. We refer to this event as the amino-terminal drop-off-reinitiation. We also showed that EF-G and RRF are involved in disassembling such an aberrant ribosome complex bearing inactive fMet-tRNAini Thereby EF-G and RRF are able to exclude unfavorable codon-anticodon combinations with weaker base pairs and alleviate the amino-terminal drop-off-reinitiation.

[Construction of EF-G knockdown strain of Mycobacterium smegmatis and drug resistance analysis].

As the only translational factor that plays a critical role in two translational processes (elongation and ribosome regeneration), GTPase elongation factor G (EF-G) is a potential target for antimicrobial agents. Both Mycobacterium smegmatis and Mycobacterium tuberculosis have two EF-G homologous coding genes, MsmEFG1 (MSMEG_1400) and MsmEFG2 (MSMEG_6535), fusA1 (Rv0684) and fusA2 (Rv0120c), respectively. MsmEFG1 (MSMEG_1400) and fusA1 (Rv0684) were identified as essential genes for bacterial growth by gene mutation library and bioinformatic analysis. To investigate the biological function and characteristics of EF-G in mycobacterium, two induced EF-G knockdown strains (Msm-ΔEFG1(KD) and Msm-ΔEFG2(KD)) from Mycobacterium smegmatis were constructed by clustered regularly interspaced short palindromic repeats interference (CRISPRi) technique. EF-G2 knockdown had no effect on bacterial growth, while EF-G1 knockdown significantly retarded the growth of mycobacterium, weakened the film-forming ability, changed the colony morphology, and increased the length of mycobacterium. It was speculated that EF-G might be involved in the division of bacteria. Minimal inhibitory concentration assay showed that inhibition of EF-G1 expression enhanced the sensitivity of mycobacterium to rifampicin, isoniazid, erythromycin, fucidic acid, capreomycin and other antibacterial agents, suggesting that EF-G1 might be a potential target for screening anti-tuberculosis drugs in the future.

Construction of EF-G knockdown strain of Mycobacterium smegmatis and drug resistance analysis / 生物工程学报

As the only translational factor that plays a critical role in two translational processes (elongation and ribosome regeneration), GTPase elongation factor G (EF-G) is a potential target for antimicrobial agents. Both Mycobacterium smegmatis and Mycobacterium tuberculosis have two EF-G homologous coding genes, MsmEFG1 (MSMEG_1400) and MsmEFG2 (MSMEG_6535), fusA1 (Rv0684) and fusA2 (Rv0120c), respectively. MsmEFG1 (MSMEG_1400) and fusA1 (Rv0684) were identified as essential genes for bacterial growth by gene mutation library and bioinformatic analysis. To investigate the biological function and characteristics of EF-G in mycobacterium, two induced EF-G knockdown strains (Msm-ΔEFG1(KD) and Msm-ΔEFG2(KD)) from Mycobacterium smegmatis were constructed by clustered regularly interspaced short palindromic repeats interference (CRISPRi) technique. EF-G2 knockdown had no effect on bacterial growth, while EF-G1 knockdown significantly retarded the growth of mycobacterium, weakened the film-forming ability, changed the colony morphology, and increased the length of mycobacterium. It was speculated that EF-G might be involved in the division of bacteria. Minimal inhibitory concentration assay showed that inhibition of EF-G1 expression enhanced the sensitivity of mycobacterium to rifampicin, isoniazid, erythromycin, fucidic acid, capreomycin and other antibacterial agents, suggesting that EF-G1 might be a potential target for screening anti-tuberculosis drugs in the future.

Crystal Structure of Mycobacterium tuberculosis Elongation Factor G1.

Mycobacterium tuberculosis (Mtb) caused an estimated 10 million cases of tuberculosis and 1.2 million deaths in 2019 globally. The increasing emergence of multidrug-resistant and extensively drug-resistant Mtb is becoming a public health threat worldwide and makes the identification of anti-Mtb drug targets urgent. Elongation factor G (EF-G) is involved in tRNA translocation on ribosomes during protein translation. Therefore, EF-G is a major focus of structural analysis and a valuable drug target of antibiotics. However, the crystal structure of Mtb EF-G1 is not yet available, and this has limited the design of inhibitors. Here, we report the crystal structure of Mtb EF-G1 in complex with GDP. The unique crystal form of the Mtb EF-G1-GDP complex provides an excellent platform for fragment-based screening using a crystallographic approach. Our findings provide a structure-based explanation for GDP recognition, and facilitate the identification of EF-G1 inhibitors with potential interest in the context of drug discovery.

Perturbation of ribosomal subunit dynamics by inhibitors of tRNA translocation.

Many antibiotics that bind to the ribosome inhibit translation by blocking the movement of tRNAs and mRNA or interfering with ribosome dynamics, which impairs the formation of essential translocation intermediates. Here we show how translocation inhibitors viomycin (Vio), neomycin (Neo), paromomycin (Par), kanamycin (Kan), spectinomycin (Spc), hygromycin B (HygB), and streptomycin (Str, an antibiotic that does not inhibit tRNA movement), affect principal motions of the small ribosomal subunits (SSU) during EF-G-promoted translocation. Using ensemble kinetics, we studied the SSU body domain rotation and SSU head domain swiveling in real time. We show that although antibiotics binding to the ribosome can favor a particular ribosome conformation in the absence of EF-G, their kinetic effect on the EF-G-induced transition to the rotated/swiveled state of the SSU is moderate. The antibiotics mostly inhibit backward movements of the SSU body and/or the head domains. Vio, Spc, and high concentrations of Neo completely inhibit the backward movements of the SSU body and head domain. Kan, Par, HygB, and low concentrations of Neo slow down both movements, but their sequence and coordination are retained. Finally, Str has very little effect on the backward rotation of the SSU body domain, but retards the SSU head movement. The data underscore the importance of ribosome dynamics for tRNA-mRNA translocation and provide new insights into the mechanism of antibiotic action.

Optimization of a fluorescent-mRNA based real-time assay for precise kinetic measurements of ribosomal translocation.

Kinetic characterization of ribosomal translocation is important for understanding the mechanism of elongation in protein synthesis. Here we have optimized a popular fluorescent-mRNA based translocation assay conducted in stopped-flow, by calibrating it with the functional tripeptide formation assay in quench-flow. We found that a fluorescently labelled mRNA, ten bases long from position +1 (mRNA+10), is best suited for both assays as it forms tripeptide at a fast rate equivalent to the longer mRNAs, and yet produces a large fluorescence change upon mRNA movement. Next, we compared the commonly used peptidyl tRNA analog, N-acetyl-Phe-tRNAPhe, with the natural dipeptidyl fMet-Phe-tRNAPhe in the stopped-flow assay. This analog translocates about two times slower than the natural dipeptidyl tRNA and produces biphasic kinetics. The rates reduce further at lower temperatures and with higher Mg2+ concentration, but improve with higher elongation factor G (EF-G) concentration, which increase both rate and amplitude of the fast phase significantly. In summary, we present here an improved real time assay for monitoring mRNA-translocation with the natural- and an N-Ac-analog of dipeptidyl tRNA.

Multifaceted Mechanism of Amicoumacin A Inhibition of Bacterial Translation.

Amicoumacin A (Ami) halts bacterial growth by inhibiting the ribosome during translation. The Ami binding site locates in the vicinity of the E-site codon of mRNA. However, Ami does not clash with mRNA, rather stabilizes it, which is relatively unusual and implies a unique way of translation inhibition. In this work, we performed a kinetic and thermodynamic investigation of Ami influence on the main steps of polypeptide synthesis. We show that Ami reduces the rate of the functional canonical 70S initiation complex (IC) formation by 30-fold. Additionally, our results indicate that Ami promotes the formation of erroneous 30S ICs; however, IF3 prevents them from progressing towards translation initiation. During early elongation steps, Ami does not compromise EF-Tu-dependent A-site binding or peptide bond formation. On the other hand, Ami reduces the rate of peptidyl-tRNA movement from the A to the P site and significantly decreases the amount of the ribosomes capable of polypeptide synthesis. Our data indicate that Ami progressively decreases the activity of translating ribosomes that may appear to be the main inhibitory mechanism of Ami. Indeed, the use of EF-G mutants that confer resistance to Ami (G542V, G581A, or ins544V) leads to a complete restoration of the ribosome functionality. It is possible that the changes in translocation induced by EF-G mutants compensate for the activity loss caused by Ami.

Synthesis of Fusidic Acid Derivatives Yields a Potent Antibiotic with an Improved Resistance Profile.

Fusidic acid (FA) is a potent steroidal antibiotic that has been used in Europe for more than 60 years to treat a variety of infections caused by Gram-positive pathogens. Despite its clinical success, FA requires significantly elevated dosing (3 g on the first day, 1.2 g on subsequent days) to minimize resistance, as FA displays a high resistance frequency, and a large shift in minimum inhibitory concentration is observed for resistant bacteria. Despite efforts to improve on these aspects, all previously constructed derivatives of FA have worse antibacterial activity against Gram-positive bacteria than the parent natural product. Here, we report the creation of a novel FA analogue that has equivalent potency against clinical isolates of Staphylococcus aureus (S. aureus) and Enterococcus faecium (E. faecium) as well as an improved resistance profile in vitro when compared to FA. Importantly, this new compound displays efficacy against an FA-resistant strain of S. aureus in a soft-tissue murine infection model. This work delineates the structural features of FA necessary for potent antibiotic activity and demonstrates that the resistance profile can be improved for this scaffold and target.

Mutations in domain IV of elongation factor EF-G confer -1 frameshifting.

A recent crystal structure of a ribosome complex undergoing partial translocation in the absence of elongation factor EF-G showed disruption of codon-anticodon pairing and slippage of the reading frame by -1, directly implicating EF-G in preservation of the translational reading frame. Among mutations identified in a random screen for dominant-lethal mutations of EF-G were a cluster of six that map to the tip of domain IV, which has been shown to contact the codon-anticodon duplex in trapped translocation intermediates. In vitro synthesis of a full-length protein using these mutant EF-Gs revealed dramatically increased -1 frameshifting, providing new evidence for a role for domain IV of EF-G in maintaining the reading frame. These mutations also caused decreased rates of mRNA translocation and rotational movement of the head and body domains of the 30S ribosomal subunit during translocation. Our results are in general agreement with recent findings from Rodnina and coworkers based on in vitro translation of an oligopeptide using EF-Gs containing mutations at two positions in domain IV, who found an inverse correlation between the degree of frameshifting and rates of translocation. Four of our six mutations are substitutions at positions that interact with the translocating tRNA, in each case contacting the RNA backbone of the anticodon loop. We suggest that EF-G helps to preserve the translational reading frame by preventing uncoupled movement of the tRNA through these contacts; a further possibility is that these interactions may stabilize a conformation of the anticodon that favors base-pairing with its codon.

The hibernating 100S complex is a target of ribosome-recycling factor and elongation factor G in Staphylococcus aureus.

The formation of translationally inactive 70S dimers (called 100S ribosomes) by hibernation-promoting factor is a widespread survival strategy among bacteria. Ribosome dimerization is thought to be reversible, with the dissociation of the 100S complexes enabling ribosome recycling for participation in new rounds of translation. The precise pathway of 100S ribosome recycling has been unclear. We previously found that the heat-shock GTPase HflX in the human pathogen Staphylococcus aureus is a minor disassembly factor. Cells lacking hflX do not accumulate 100S ribosomes unless they are subjected to heat exposure, suggesting the existence of an alternative pathway during nonstressed conditions. Here, we provide biochemical and genetic evidence that two essential translation factors, ribosome-recycling factor (RRF) and GTPase elongation factor G (EF-G), synergistically split 100S ribosomes in a GTP-dependent but tRNA translocation-independent manner. We found that although HflX and the RRF/EF-G pair are functionally interchangeable, HflX is expressed at low levels and is dispensable under normal growth conditions. The bacterial RRF/EF-G pair was previously known to target only the post-termination 70S complexes; our results reveal a new role in the reversal of ribosome hibernation that is intimately linked to bacterial pathogenesis, persister formation, stress responses, and ribosome integrity.