Peptidyl Transferase Center Research Articles

Structural Basis for Linezolid Binding Site Rearrangement in the Staphylococcus aureus Ribosome.

ABSTRACTAn unorthodox, surprising mechanism of resistance to the antibiotic linezolid was revealed by cryo-electron microscopy (cryo-EM) in the 70S ribosomes from a clinical isolate of Staphylococcus aureus. This high-resolution structural information demonstrated that a single amino acid deletion in ribosomal protein uL3 confers linezolid resistance despite being located 24 Å away from the linezolid binding pocket in the peptidyl-transferase center. The mutation induces a cascade of allosteric structural rearrangements of the rRNA that ultimately results in the alteration of the antibiotic binding site.

Effect of Nascent Peptide Steric Bulk on Elongation Kinetics in the Ribosome Exit Tunnel

All proteins are synthesized by the ribosome, a macromolecular complex that accomplishes the life-sustaining tasks of faithfully decoding mRNA and catalyzing peptide bond formation at the peptidyl transferase center (PTC). The ribosome has evolved an exit tunnel to host the elongating new peptide, protect it from proteolytic digestion, and guide its emergence. It is here that the nascent chain begins to fold. This folding process depends on the rate of translation at the PTC. We report here that besides PTC events, translation kinetics depend on steric constraints on nascent peptide side chains and that confined movements of cramped side chains within and through the tunnel fine-tune elongation rates.

Uncovering the Cellular Target of Agelastatin A

McClary etal. (2017) identify the eukaryotic ribosome as a cellular target of agelastatin A, resolving the long-standing mystery surrounding the cytotoxic natural product's mechanism of action. Structural and modeling studies further pinpointed the molecule's binding site to the ribosome peptidyl transferase center, revealing key molecular interactions that drive binding.

Ribosomal proteins as documents of the transition from unstructured (poly)peptides to folded proteins.

For the most part, contemporary proteins can be traced back to a basic set of a few thousand domain prototypes, many of which were already established in the Last Universal Common Ancestor of life on Earth, around 3.5 billion years ago. The origin of these domain prototypes, however, remains poorly understood. One hypothesis posits that they arose from an ancestral set of peptides, which acted as cofactors of RNA-mediated catalysis and replication. Initially, these peptides were entirely dependent on the RNA scaffold for their structure, but as their complexity increased, they became able to form structures by excluding water through hydrophobic contacts, making them independent of the RNA scaffold. Their ability to fold was thus an emergent property of peptide-RNA coevolution. The ribosome is the main survivor of this primordial RNA world and offers an excellent model system for retracing the steps that led to the folded proteins of today, due to its very slow rate of change. Close to the peptidyl transferase center, which is the oldest part of the ribosome, proteins are extended and largely devoid of secondary structure; further from the center, their secondary structure content increases and supersecondary topologies become common, although the proteins still largely lack a hydrophobic core; at the ribosomal periphery, supersecondary structures coalesce around hydrophobic cores, forming folds that resemble those seen in proteins of the cytosol. Collectively, ribosomal proteins thus offer a window onto the time when proteins were acquiring the ability to fold.

Peptidyl Transferase Center and the Emergence of the Translation System.

In this work, the three-dimensional (3D) structure of the ancestral Peptidyl Transferase Center (PTC) built by concatamers of ancestral sequences of tRNAs was reconstructed, and its possible interactions with tRNAs molecules were analyzed. The 3D structure of the ancestral PTC was also compared with the current PTC of T. thermophilus. Docking experiments between the ancestral PTC and tRNAs suggest that in the origin of the translation system, the PTC functioned as an adhesion center for tRNA molecules. The approximation of tRNAs charged with amino acids to the PTC permitted peptide synthesis without the need of a genetic code.

Unraveling The Role of Helicases in Nonfunctional rRNA Decay

rRNAs are essential, long‐lived, and highly abundant, comprising up to 90% of total cellular RNA. However, when structurally intact rRNAs in Saccharomyces cerevisiae are functionally compromised, they are instead rapidly degraded through a quality control pathway termed nonfunctional ribosomal RNA decay (NRD). Currently, two mechanistically distinct types of NRD are known: 18S NRD and 25S NRD. 18S NRD is translation dependent and removes rRNAs with loss of function mutations in the small subunit decoding site. The 18S NRD mechanism involves the translation factor‐like protein Dom34 and its binding partner Hbs1. On the other hand, 25S NRD, which occurs prior to subunit joining, is translation independent and removes nonfunctional rRNAs containing mutations in the large subunit peptidyl transferase center. The 25S NRD mechanism involves polyubiquination of proteins associated with a nonfunctional 25S rRNA through the E3 ligase components Rtt101 and Mms1. While some of the proteins that are involved in either 18S or 25S NRD are known, we have not yet elucidated the complete molecular mechanism of NRD. Since RNA helicases have been implicated in other RNA degradation pathways, we hypothesize that one or more helicases may be important for either or both NRD mechanisms. S. cerevisiae contains 44 different helicases, 14 of which are non‐essential. Thus, we are initially investigating whether any of these 14 helicases are involved in NRD using single gene deletion yeast strains. We transformed BY4741 and the 14 non‐essential helicase knock‐out strains with various rRNA expression plasmids that encode for functional and/or nonfunctional versions of the 18S and 25S rRNAs. These plasmid‐derived rRNAs also contain short, unique sequence tags that allows for specific detection of these rRNAs in the presence of total cellular rRNA. We measured the growth phenotypes of these transformed strains at various temperatures in liquid cultures. At standard temperatures, we see a slow growth phenotype in some of the deletion strains transformed with either nonfunctional 18S or 25S rRNA expression plasmids. Since some helicase deletion strains exhibit either cold‐ or temperature‐sensitivities, we are currently measuring growth phenotypes at these normally restrictive temperatures. In addition, we are measuring the rRNA expression levels in these deletion strains using an RT‐qPCR assay to quantify the level of NRD activity. We hope this work will provide insight into the role of helicases in RNA degradation mechanisms and contribute to our understanding of NRD.Support or Funding InformationThis work is supported by the Howard Hughes Medical Institute.

Critical 23S rRNA interactions for macrolide-dependent ribosome stalling on the ErmCL nascent peptide chain.

The nascent peptide exit tunnel has recently been identified as a functional region of ribosomes contributing to translation regulation and co-translational protein folding. Inducible expression of the erm resistance genes depends on ribosome stalling at specific codons of an upstream open reading frame in the presence of an exit tunnel-bound macrolide antibiotic. The molecular basis for this translation arrest is still not fully understood. Here, we used a nucleotide analog interference approach to unravel important functional groups on 23S rRNA residues in the ribosomal exit tunnel for ribosome stalling on the ErmC leader peptide. By replacing single nucleobase functional groups or even single atoms we were able to demonstrate the importance of A2062, A2503 and U2586 for drug-dependent ribosome stalling. Our data show that the universally conserved A2062 and A2503 are capable of forming a non-Watson–Crick base pair that is critical for sensing and transmitting the stalling signal from the exit tunnel back to the peptidyl transferase center of the ribosome. The nucleobases of A2062, A2503 as well as U2586 do not contribute significantly to the overall mechanism of protein biosynthesis, yet their elaborate role for co-translational monitoring of nascent peptide chains inside the exit tunnel can explain their evolutionary conservation.

Mechanism of ribosome rescue by ArfA and RF2.

ArfA rescues ribosomes stalled on truncated mRNAs by recruiting release factor RF2, which normally binds stop codons to catalyze peptide release. We report two 3.2 Å resolution cryo-EM structures - determined from a single sample - of the 70S ribosome with ArfA•RF2 in the A site. In both states, the ArfA C-terminus occupies the mRNA tunnel downstream of the A site. One state contains a compact inactive RF2 conformation. Ordering of the ArfA N-terminus in the second state rearranges RF2 into an extended conformation that docks the catalytic GGQ motif into the peptidyl-transferase center. Our work thus reveals the structural dynamics of ribosome rescue. The structures demonstrate how ArfA 'senses' the vacant mRNA tunnel and activates RF2 to mediate peptide release without a stop codon, allowing stalled ribosomes to be recycled.

Dual effect of chloramphenicol peptides on ribosome inhibition.

Chloramphenicol peptides were recently established as useful tools for probing nascent polypeptide chain interaction with the ribosome, either biochemically, or structurally. Here, we present a new 10mer chloramphenicol peptide, which exerts a dual inhibition effect on the ribosome function affecting two distinct areas of the ribosome, namely the peptidyl transferase center and the polypeptide exit tunnel. According to our data, the chloramphenicol peptide bound on the chloramphenicol binding site inhibits the formation of both acetyl-phenylalanine-puromycin and acetyl-lysine-puromycin, showing, however, a decreased peptidyl transferase inhibition compared to chloramphenicol-mediated inhibition per se. Additionally, we found that the same compound is a strong inhibitor of green fluorescent protein synthesis in a coupled in vitro transcription-translation assay as well as a potent inhibitor of lysine polymerization in a poly(A)-programmed ribosome, showing that an additional inhibitory effect may exist. Since chemical protection data supported the interaction of the antibiotic with bases A2058 and A2059 near the entrance of the tunnel, we concluded that the extra inhibition effect on the synthesis of longer peptides is coming from interactions of the peptide moiety of the drug with residues comprising the ribosomal tunnel, and by filling up the tunnel and blocking nascent chain progression through the restricted tunnel. Therefore, the dual interaction of the chloramphenicol peptide with the ribosome increases its inhibitory effect and opens a new window for improving the antimicrobial potency of classical antibiotics or designing new ones.

Sequestration of Ribosome during Protein Aggregate Formation: Contribution of ribosomal RNA

An understanding of the mechanisms underlying protein aggregation and cytotoxicity of the protein aggregates is crucial in the prevention of several diseases in humans. Ribosome, the cellular protein synthesis machine is capable of acting as a protein folding modulator. The peptidyltransferase center residing in the domain V of large ribosomal subunit 23S rRNA is the centre for the protein folding ability of the ribosome and is also the cellular target of several antiprion compounds. Our in vitro studies unexpectedly reveal that the partial unfolding or aggregation of lysozyme under reducing conditions in presence of the ribosome can induce aggregation of ribosomal components. Electrostatic interactions complemented by specific rRNA-protein interaction drive the ribosome-protein aggregation process. Under similar conditions the rRNA, especially the large subunit rRNA and in vitro transcribed RNA corresponding to domain V of 23S rRNA (bDV RNA) stimulates lysozyme aggregation leading to RNA-protein aggregate formation. Protein aggregation during the refolding of non-disulfide containing protein BCAII at high concentrations also induces ribosome aggregation. BCAII aggregation was also stimulated in presence of the large subunit rRNA. Our observations imply that the specific sequestration of the translation machine by aggregating proteins might contribute to their cytotoxicity.

Structural snapshot of cytoplasmic pre-60S ribosomal particles bound by Nmd3, Lsg1, Tif6 and Reh1.

A key step in ribosome biogenesis is the nuclear export of pre-ribosomal particles. Nmd3, a highly conserved protein in eukaryotes, is a specific adaptor required for the export of pre-60S particles. Here we used cryo-electron microscopy (cryo-EM) to characterize Saccharomyces cerevisiae pre-60S particles purified with epitope-tagged Nmd3. Our structural analysis indicates that these particles belong to a specific late stage of cytoplasmic pre-60S maturation in which ribosomal proteins uL16, uL10, uL11, eL40 and eL41 are deficient, but ribosome assembly factors Nmd3, Lsg1, Tif6 and Reh1 are present. Nmd3 and Lsg1 are located near the peptidyl-transferase center (PTC). In particular, Nmd3 recognizes the PTC in its near-mature conformation. In contrast, Reh1 is anchored to the exit of the polypeptide tunnel, with its C terminus inserted into the tunnel. These findings pinpoint a structural checkpoint role for Nmd3 in PTC assembly, and provide information about functional and mechanistic roles of these assembly factors in the maturation of the 60S ribosomal subunit.

The assembly factor Erb1 functions in multiple remodeling events during 60S ribosomal subunit assembly in S. cerevisiae.

A major gap in our understanding of ribosome assembly is knowing the precise function of each of the ∼200 assembly factors. The steps in subunit assembly in which these factors participate have been examined for the most part by depleting each protein from cells. Depletion of the assembly factor Erb1 prevents stable assembly of seven other interdependent assembly factors with pre-60S subunits, resulting in turnover of early preribosomes, before the ITS1 spacer can be removed from 27SA3 pre-rRNA. To investigate more specific functions of Erb1, we constructed eight internal deletions of 40–60 amino acid residues each, spanning the amino-terminal half of Erb1. The erb1Δ161–200 and erb1Δ201–245 deletion mutations block a later step than depletion of Erb1, namely cleavage of the C2 site that initiates removal of the ITS2 spacer. Two other remodeling events fail to occur in these erb1 mutants: association of twelve different assembly factors with domain V of 25S rRNA, including the neighborhood surrounding the peptidyl transferase center, and stable association of ribosomal proteins with rRNA surrounding the polypeptide exit tunnel. This suggests that successful initiation of construction of these functional centers is a checkpoint for committing to spacer removal.

Efficacy of Lefamulin Against Staphylococcus aureus-Induced Bacteremia in a Neutropenic and Immunocompetent Murine Model

Background S. aureus (SA) is a major human pathogen that causes invasive, clinical infections including bacteremia. Lefamulin (LEF) is the first semi-synthetic, pleuromutilin antibiotic for IV and oral use in humans. LEF is currently in Phase 3 trials for the treatment of community-acquired bacterial pneumonia (CABP). LEF specifically inhibits bacterial protein synthesis by binding to the peptidyl transferase center (PTC) via four H-bonds and other interactions at the A- and P-site resulting in an “induced fit.” LEF has been shown to be highly active against bacterial pathogens causing bacteremia, including SA. This study investigated the efficacy of LEF and comparators against SA in a neutropenic and immunocompetent murine bacteremia model.MethodsExperimentally induced MSSA bacteremia (inoculum ~2 × 107 CFU/mouse) was established in immunocompromised and immunocompetent mice. Infected mice received a single subcutaneous dose of either LEF or comparator (Table 1) 1 hours post-inoculation, mimicking human therapeutic exposures. A control group of infected mice were sacrificed directly before treatment to establish a baseline CFU count and comparison with the bacterial load of treated animals 24 hours post drug administration.ResultsIrrespective of the immune status, LEF showed superior efficacy to linezolid (LZD) and tigecycline (TGC) against MSSA, reducing the bacterial burden more than 4 log10 CFU/mL within 24 hours (Table 1). A comparable reduction of bacterial burden was observed between LEF and daptomycin (DAP) or vancomycin (VAN) treatment.ConclusionLEF showed comparable therapeutic outcome to DAP or VAN in this acute experimental infection model, while showing superior killing as compared with LZD or TGC. The efficacy of LEF was maintained under neutropenic conditions with >4log10 ΔCFU/mL at clinically relevant exposures. This study supports continued evaluation of LEF for as a potential treatment of staphylococcal bacteremia.Disclosures E. Fischer, Nabriva Therapeutics AG: Employee and Shareholder, Salary; B. C. Kappes, Nabriva Therapeutics AG: Employee and Shareholder, Salary; W. W. Wicha, Nabriva Therapeutics AG: Employee and Shareholder, Salary

DbpA is a region-specific RNA helicase.

DbpA is a DEAD-box RNA helicase implicated in RNA structural rearrangements in the peptidyl transferase center. DbpA contains an RNA binding domain, responsible for tight binding of DbpA to hairpin 92 of 23S ribosomal RNA, and a RecA-like catalytic core responsible for double-helix unwinding. It is not known if DbpA unwinds only the RNA helices that are part of a specific RNA structure, or if DbpA unwinds any RNA helices within the catalytic core's grasp. In other words, it is not known if DbpA is a site-specific enzyme or region-specific enzyme. In this study, we used protein and RNA engineering to investigate if DbpA is a region-specific or a site-specific enzyme. Our data suggest that DbpA is a region-specific enzyme. This conclusion has an important implication for the physiological role of DbpA. It suggests that during ribosome assembly, DbpA could bind with its C-terminal RNA binding domain to hairpin 92, while its catalytic core may unwind any double-helices in its vicinity. The only requirement for a double-helix to serve as a DbpA substrate is for the double-helix to be positioned within the catalytic core's grasp.

Structural basis for ArfA-RF2-mediated translation termination on mRNAs lacking stop codons.

In bacteria, ribosomes stalled on truncated mRNAs that lack a stop codon are rescued by the transfer-messenger RNA (tmRNA), alternative rescue factor A (ArfA) or ArfB systems. Although tmRNA-ribosome and ArfB-ribosome structures have been determined, how ArfA recognizes the presence of truncated mRNAs and recruits the canonical termination release factor RF2 to rescue the stalled ribosomes is unclear. Here we present a cryo-electron microscopy reconstruction of the Escherichia coli 70S ribosome stalled on a truncated mRNA in the presence of ArfA and RF2. The structure shows that the C terminus of ArfA binds within the mRNA entry channel on the small ribosomal subunit, and explains how ArfA distinguishes between ribosomes that bear truncated or full-length mRNAs. The N terminus of ArfA establishes several interactions with the decoding domain of RF2, and this finding illustrates how ArfA recruits RF2 to the stalled ribosome. Furthermore, ArfA is shown to stabilize a unique conformation of the switch loop of RF2, which mimics the canonical translation termination state by directing the catalytically important GGQ motif within domain 3 of RF2 towards the peptidyl-transferase centre of the ribosome. Thus, our structure reveals not only how ArfA recruits RF2 to the ribosome but also how it promotes an active conformation of RF2 to enable translation termination in the absence of a stop codon.

Synthesis and antibacterial activities of novel pleuromutilin derivatives with a substituted pyrimidine moiety

The alarming growth of multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) has become a major global health hazard. Therefore, urgent demand for new antibiotics with a unique mechanism of action is very necessary. The present study reports the design, synthesis, and antibacterial studies of a series of novel pleuromutilin derivatives with substituted 6-amino pyrimidine moieties. Most of the tested compounds exhibited highly potent anti-MRSA or Staphylococcus aureus (S. aureus) activities. 14-O-[(4,6-Diamino -pyrimidine-2-yl) thioacetyl] mutilin (3) and 14-O-[(2-((3R)-3-Hydroxymethylpiperidine-1-yl)-acetamido-6-aminopyrimidine-2-yl) thioacetyl] mutilin (5h) were the most active compounds and showed higher antibacterial activities. Compound 3 displayed rapid bactericidal activity and affected bacterial growth with the same manner as tiamulin fumarate. Docking experiments for compounds 3 and 5h carried out on the peptidyl transferase center (PTC) of 23S rRNA provided the information about the binding model. In vivo mouse systemic infection experimental results confirmed the therapeutic efficacy of compound 3, with ED50 of 4.22 mg/kg body weight against MRSA.

Inhibition of Escherichia coli ribosome subunit dissociation by chloramphenicol and Blasticidin: a new mode of action of the antibiotics.

Ribosome synthesizes protein in all organisms and is the target for multiple antimicrobial agents. Our study demonstrates that chloramphenicol and blasticidin S that target the peptidyl transferase centre of the bacterial ribosome can then inhibit dissociation of 70S ribosome mediated by (i) unfolded protein, (ii) translation factors or (iii) low Mg+2 concentrations invitro and thereby suppresses ribosomal degradation during carbon starvation invivo. The demonstration of this new mode of action furthers the understanding of these broad-spectrum antibiotics that differentially inhibit protein synthesis in prokaryotic and eukaryotic cells.

Induced fit of the peptidyl-transferase center of the ribosome and conformational freedom of the esterified amino acids.

The catalytic site of most enzymes can efficiently handle only one substrate. In contrast, the ribosome is capable of polymerizing at a similar rate at least 20 different kinds of amino acids from aminoacyl-tRNA carriers while using just one catalytic site, the peptidyl-transferase center (PTC). An induced-fit mechanism has been uncovered in the PTC, but a possible connection between this mechanism and the uniform handling of the substrates has not been investigated. We present an analysis of published ribosome structures supporting the hypothesis that the induced fit eliminates unreactive rotamers predominantly populated for some A-site aminoacyl esters before induction. We show that this hypothesis is fully consistent with the wealth of kinetic data obtained with these substrates. Our analysis reveals that induction constrains the amino acids into a reactive conformation in a side-chain independent manner. It allows us to highlight the rationale of the PTC structural organization, which confers to the ribosome the very unusual ability to handle large as well as small substrates.

Peptide Release on the Ribosome Involves Substrate-Assisted Base Catalysis

Termination of protein synthesis on the ribosome involves hydrolysis of the ester bond between the P-site tRNA and the nascent peptide chain. This reaction occurs in the peptidyl transferase center and is triggered by the class I release factors RF1 and RF2 in prokaryotes. Peptidyl-tRNA hydrolysis is pH-dependent, and experimental results suggest that an ionizable group with pKa > 9 is involved in the reaction. The nature of this group is, however, unknown. To resolve this problem, we conducted density functional theory calculations using a large cluster model of the peptidyl transferase center. Our calculations reveal that peptidyl-tRNA hydrolysis occurs via a base-catalyzed mechanism with a predicted activation energy of 15.8 kcal mol–1, which is in good agreement with experimental data. In this mechanism, the P-site A76 2′-OH group is deprotonated and acts as the general base by activating the nucleophilic water molecule. The energy cost of deprotonating the 2′-hydroxyl group at pH 7.5 is estimated to ...

Quantitative determination of ribosome nascent chain stability

Accurate protein folding is essential for proper cellular and organismal function. In the cell, protein folding is carefully regulated; changes in folding homeostasis (proteostasis) can disrupt many cellular processes and have been implicated in various neurodegenerative diseases and other pathologies. For many proteins, the initial folding process begins during translation while the protein is still tethered to the ribosome; however, most biophysical studies of a protein's energy landscape are carried out in isolation under idealized, dilute conditions and may not accurately report on the energy landscape in vivo. Thus, the energy landscape of ribosome nascent chains and the effect of the tethered ribosome on nascent chain folding remain unclear. Here we have developed a general assay for quantitatively measuring the folding stability of ribosome nascent chains, and find that the ribosome exerts a destabilizing effect on the polypeptide chain. This destabilization decreases as a function of the distance away from the peptidyl transferase center. Thus, the ribosome may add an additional layer of robustness to the protein-folding process by avoiding the formation of stable partially folded states before the protein has completely emerged from the ribosome.