Biology subjects

Syroegin, E. A.

Publications and source records attributed to Syroegin, E. A..

6 recordsLinked to original sources

bioRxiv2025

Structural insights into context-specific inhibition of bacterial translation by macrolides

The ribosomes peptidyl transferase center (PTC) catalyzes peptide bond formation during protein synthesis and is targeted by many antibiotic classes. Remarkably, macrolides that bind in the peptide exit tunnel some [~]10[A] away from the PTC also remotely inhibit PTC and cause translational arrest depending on the synthesized polypeptide sequence. The Arg/Lys-X-Arg/Lys (also known as +X+) motif is particularly susceptible to this inhibition, as peptidyl-tRNA carrying nascent peptide with penultimate arginine or lysine residue fails to react with aminoacyl-tRNA carrying the same amino acids in the presence of macrolides. While structural studies of macrolide-bound ribosomes have shed light on the context-specific nature of this inhibition, the precise roles of the drug, ribosome, and tRNA in modulating PTC activity remain unclear. In this study, we present a detailed structural analysis of ribosome-nascent chain complexes (RNCs) that represent either arrested or non-arrested states, containing various combinations of peptidyl- and aminoacyl-tRNAs, with or without macrolides. Our findings reveal a dynamic interaction between the ribosome-bound drug, the nascent peptide, and the incoming amino acid, which collectively modulates PTC function. This lays the foundation for designing antibiotics that can overcome drug resistance by preventing the induction of inducible erm genes in pathogens.

molecular biology↗

bioRxiv2024

Toxic Small Alarmone Synthetase FaRel2 inhibits translation by pyrophosphorylating tRNAGly and tRNAThr

Translation-targeting toxic Small Alarmone Synthetases (toxSAS) are effectors of bacterial Toxin-Antitoxin systems that pyrophosphorylate the 3-CCA end of tRNA to prevent aminoacylation. toxSAS are implicated in antiphage immunity: phage detection triggers the toxSAS activity to shut down viral production. We show that the toxSAS FaRel2 inspects the tRNA acceptor stem to specifically select tRNAGly and tRNAThr. The 1st, 2nd, 4th and 5th base pairs the stem act as the specificity determinants. We show that the toxSASs PhRel2 and CapRelSJ46 differ in tRNA specificity from FaRel2, and rationalise this through structural modelling: while the universal 3-CCA end slots into a highly conserved CCA recognition groove, the acceptor stem recognition region is variable across toxSAS diversity. As phages use tRNA isoacceptors to overcome tRNA-targeting defences, we hypothesise that highly evolvable modular tRNA recognition allows for the escape of viral countermeasures through tRNA substrate specificity switching.

microbiology↗

bioRxiv2024

Rationally Designed Pooled CRISPRi-seq Uncovers a Novel Inhibitor of Bacterial Peptidyl-tRNA Hydrolase

Pooled knockdown libraries of essential genes are useful tools for elucidating the mechanisms of action of antibacterial compounds, a pivotal step in antibiotic discovery. However, achieving genomic coverage of antibacterial targets poses a challenge due to the uneven proliferation of knockdown mutants during pooled growth, leading to the unintended loss of important targets. To overcome this issue, we describe the construction of CIMPLE (CRISPRi-mediated pooled library of essential genes), a rationally designed pooled knockdown library built in a model antibiotic-resistant bacteria, Burkholderia cenocepacia. By analyzing growth parameters of clonal knockdown populations of an arrayed CRISPRi library, we predicted strain depletion levels during pooled growth and adjusted mutant relative abundance, approaching genomic coverage of antibacterial targets during antibiotic exposure. We first benchmarked CIMPLE by chemical-genetic profiling of known antibacterials, then applied it to an uncharacterized bacterial growth inhibitor from a new class. CRISPRi-Seq with CIMPLE, followed by biochemical validation, revealed that the novel compound targets the peptidyl-tRNA hydrolase (Pth). Overall, CIMPLE leverages the advantages of arrayed and pooled CRISPRi libraries to uncover unexplored targets for antibiotic action. SummaryBacterial mutant libraries in which antibiotic targets are downregulated are useful tools to functionally characterize novel antimicrobials. These libraries are used for chemical-genetic profiling as target-compound interactions can be inferred by differential fitness of mutants during pooled growth. Mutants that are functionally related to the antimicrobial mode of action are usually depleted from the pool upon exposure to the drug. Although powerful, this method can fail when the unequal proliferation of mutant strains before exposure causes mutants to fall below the detection level in the library pool. To address this issue, we constructed an arrayed essential gene mutant library (EGML) in the antibiotic-resistant bacterium Burkholderia cenocepacia using CRISPR interference (CRISPRi) and analyzed the growth parameters of individual mutant strains. We then modelled depletion levels during pooled growth and used the model to rationally design an optimized CRISPR interference-mediated pooled library of essential genes (CIMPLE). By adjusting the initial inoculum of the knockdown mutants, we achieved coverage of the bacterial essential genome with mutant sensitization. We exposed CIMPLE to a recently discovered antimicrobial of a novel class and discovered it inhibits the peptidyl-tRNA hydrolase, an essential bacterial enzyme. In summary, we demonstrate the utility of CIMPLE and CRISPRi-Seq to uncover the mechanism of action of novel antimicrobial compounds. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/592284v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@b09cf9org.highwire.dtl.DTLVardef@1119fc9org.highwire.dtl.DTLVardef@116b9forg.highwire.dtl.DTLVardef@1b0ce3a_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

Structural basis of Cfr-mediated antimicrobial resistance and mechanisms for its evasion

The ribosome is an essential drug target as many classes of clinically important antibiotics bind and inhibit its functional centers. The catalytic peptidyl transferase center (PTC) is targeted by the broadest array of inhibitors belonging to several chemical classes. One of the most abundant and clinically prevalent mechanisms of resistance to PTC-acting drugs is C8-methylation of the universally conserved adenine residue 2503 (A2503) of the 23S rRNA by the methyltransferase Cfr. Despite its clinical significance, a sufficient understanding of the molecular mechanisms underlying Cfr-mediated resistance is currently lacking. In this work, we developed a method to express a functionally-active Cfr-methyltransferase in the thermophilic bacterium Thermus thermophilus and report a set of high-resolution structures of the Cfr-modified 70S ribosome containing aminoacyl- and peptidyl-tRNAs. Our structures reveal that an allosteric rearrangement of nucleotide A2062 upon Cfr-methylation of A2503 is likely responsible for the inability of some PTC inhibitors to bind to the ribosome, providing additional insights into the Cfr resistance mechanism. Lastly, by determining the structures of the Cfr-methylated ribosome in complex with the antibiotics iboxamycin and tylosin, we provide the structural bases behind two distinct mechanisms of evading Cfr-mediated resistance.

molecular biology↗

bioRxiv2022

Insights into the ribosome function from the structures of non-arrested ribosome nascent chain complexes.

During protein synthesis, the growing polypeptide chain threads through the nascent peptide exit tunnel that spans the body of the large ribosomal subunit while simultaneously acting as a modulator of ribosomal activity by itself or by sensing various small molecules, such as metabolites or antibiotics appearing in the tunnel. While arrested ribosome nascent chain complexes (RNCCs) have been extensively studied structurally, little attention has been given to the RNCCs that represent the functionally active state of the ribosome. This is in part due to the lack of a simple and reliable procedure for the large-scale preparation of peptidyl-tRNAs. Here we report a new chemoenzymatic approach based on native chemical ligation reaction for the facile synthesis of stably linked peptidyl-tRNAs that were used to determine several structures of RNCCs in the functional pre-attack state of the peptidyl transferase center (PTC) at the highest resolution available to date. These structures reveal a previously unknown role of the ribosome in stabilization of the growing polypeptide within the PTC and suggest an extended entropic trap model that mechanistically rationalizes how ribosome acts with comparable efficiencies upon a multitude of possible growing peptides having various sequences. Our structures also provide new insights into the mechanism of PTC functioning and explain what makes ribosome a versatile catalyst.

molecular biology↗

bioRxiv2021

Structural basis for the context-specific action of classic peptidyl transferase inhibitors.

Ribosome-targeting antibiotics serve both as powerful antimicrobials and as tools for studying the ribosome. The ribosomal catalytic site, the peptidyl transferase center (PTC), is targeted by a large number of various drugs. The classical and best-studied PTC-acting antibiotic chloramphenicol, as well as the newest clinically significant linezolid, were considered indiscriminate inhibitors of every round of peptide bond formation, presumably inhibiting protein synthesis by stalling ribosomes at every codon of every gene being translated. However, it was recently discovered that chloramphenicol or linezolid, and many other PTC-targeting drugs, preferentially arrest translation when the ribosome needs to polymerize particular amino acid sequences. The molecular mechanisms and structural bases that underlie this phenomenon of context-specific action of even the most basic ribosomal antibiotics, such as chloramphenicol, are unknown. Here we present high-resolution structures of ribosomal complexes, with or without chloramphenicol, carrying specific nascent peptides that support or negate the drug action. Our data suggest that specific amino acids in the nascent chains directly modulate the antibiotic affinity to the ribosome by either establishing specific interactions with the drug molecule or obstructing its placement in the binding site. The model that emerged from our studies rationalizes the critical importance of the penultimate residue of a growing peptide for the ability of the drug to stall translation and provides the first atomic-level understanding of context specificity of antibiotics that inhibit protein synthesis by acting upon the PTC.

molecular biology↗