scholarly journals Context-specific action of macrolide antibiotics on the eukaryotic ribosome

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Maxim S. Svetlov ◽  
Timm O. Koller ◽  
Sezen Meydan ◽  
Vaishnavi Shankar ◽  
Dorota Klepacki ◽  
...  
Keyword(s):  
Amino Acid Sequences ◽  
Macrolide Antibiotics ◽  
Single Mutation ◽  
Sequence Motifs ◽  
Eukaryotic Translation ◽  
Eukaryotic Ribosome ◽  
Cellular Translation ◽  
Distinct Sequence ◽  
Exit Tunnel ◽  
Context Specific

AbstractMacrolide antibiotics bind in the nascent peptide exit tunnel of the bacterial ribosome and prevent polymerization of specific amino acid sequences, selectively inhibiting translation of a subset of proteins. Because preventing translation of individual proteins could be beneficial for the treatment of human diseases, we asked whether macrolides, if bound to the eukaryotic ribosome, would retain their context- and protein-specific action. By introducing a single mutation in rRNA, we rendered yeast Saccharomyces cerevisiae cells sensitive to macrolides. Cryo-EM structural analysis showed that the macrolide telithromycin binds in the tunnel of the engineered eukaryotic ribosome. Genome-wide analysis of cellular translation and biochemical studies demonstrated that the drug inhibits eukaryotic translation by preferentially stalling ribosomes at distinct sequence motifs. Context-specific action markedly depends on the macrolide structure. Eliminating macrolide-arrest motifs from a protein renders its translation macrolide-tolerant. Our data illuminate the prospects of adapting macrolides for protein-selective translation inhibition in eukaryotic cells.

scholarly journals Structural and mechanistic basis for translation inhibition by macrolide and ketolide antibiotics

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Bertrand Beckert ◽  
Elodie C. Leroy ◽  
Shanmugapriya Sothiselvam ◽  
Lars V. Bock ◽  
Maxim S. Svetlov ◽  
...  
Keyword(s):  
Antibiotic Resistance Genes ◽  
Structural Basis ◽  
Sequence Motifs ◽  
Specific Sequence ◽  
Bacterial Ribosome ◽  
Translational Arrest ◽  
Exit Tunnel ◽  
Mechanistic Basis ◽  
Dynamics Simulations ◽  
Context Specific

AbstractMacrolides and ketolides comprise a family of clinically important antibiotics that inhibit protein synthesis by binding within the exit tunnel of the bacterial ribosome. While these antibiotics are known to interrupt translation at specific sequence motifs, with ketolides predominantly stalling at Arg/Lys-X-Arg/Lys motifs and macrolides displaying a broader specificity, a structural basis for their context-specific action has been lacking. Here, we present structures of ribosomes arrested during the synthesis of an Arg-Leu-Arg sequence by the macrolide erythromycin (ERY) and the ketolide telithromycin (TEL). Together with deep mutagenesis and molecular dynamics simulations, the structures reveal how ERY and TEL interplay with the Arg-Leu-Arg motif to induce translational arrest and illuminate the basis for the less stringent sequence-specific action of ERY over TEL. Because programmed stalling at the Arg/Lys-X-Arg/Lys motifs is used to activate expression of antibiotic resistance genes, our study also provides important insights for future development of improved macrolide antibiotics.

scholarly journals Binding Site of the Bridged Macrolides in the Escherichia coli Ribosome

2005 ◽  
Vol 49 (1) ◽  
pp. 281-288 ◽  
Author(s):  
Liqun Xiong ◽  
Yakov Korkhin ◽  
Alexander S. Mankin
Keyword(s):  
Escherichia Coli ◽  
Tight Binding ◽  
Dimethyl Sulfate ◽  
23S Rrna ◽  
Side Chain ◽  
Macrolide Antibiotics ◽  
Side Chains ◽  
Alkyl Aryl ◽  
E Coli ◽  
Exit Tunnel

ABSTRACT Ketolides represent the latest group of macrolide antibiotics. Tight binding of ketolides to the ribosome appears to correlate with the presence of an extended alkyl-aryl side chain. Recently developed 6,11-bridged bicyclic ketolides extend the spectrum of platforms used to generate new potent macrolides with extended alkyl-aryl side chains. The purpose of the present study was to characterize the site of binding and the action of bridged macrolides in the ribosomes of Escherichia coli. All the bridged macrolides investigated efficiently protected A2058 and A2059 in domain V of 23S rRNA from modification by dimethyl sulfate and U2609 from modification by carbodiimide. In addition, bridged macrolides that carry extended alkyl-aryl side chains protruding from the 6,11 bridge protected A752 in helix 35 of domain II of 23S rRNA from modification by dimethyl sulfate. Bridged macrolides efficiently displaced erythromycin from the ribosome in a competition binding assay. The A2058G mutation in 23S rRNA conferred resistance to the bridged macrolides. The U2609C mutation, which renders E. coli resistant to the previously studied ketolides telithromycin and cethromycin, barely affected cell susceptibility to the bridged macrolides used in this study. The results of the biochemical and genetic studies indicate that in the E. coli ribosome, bridged macrolides bind in the nascent peptide exit tunnel at the site previously described for other macrolide antibiotics. The presence of the side chain promotes the formation of specific interactions with the helix 35 of 23S rRNA.

scholarly journals Potential Selection of LMP1 Variants in Nasopharyngeal Carcinoma

2004 ◽  
Vol 78 (2) ◽  
pp. 868-881 ◽  
Author(s):  
Rachel H. Edwards ◽  
Diane Sitki-Green ◽  
Dominic T. Moore ◽  
Nancy Raab-Traub
Keyword(s):  
Amino Acid ◽  
Cytotoxic T Lymphocyte ◽  
Amino Acid Sequences ◽  
Barr Virus ◽  
Distinct Sequence ◽  
Epstein Barr ◽  
Virus Latent Membrane Protein ◽  
Multiple Strains ◽  
Carboxy Terminal ◽  
Selection Of

ABSTRACT Seven distinct sequence variants of the Epstein-Barr virus latent membrane protein 1 (LMP1) have been identified by distinguishing amino acid changes in the carboxy-terminal domain. In this study the transmembrane domains are shown to segregate identically with the distinct carboxy-terminal amino acid sequences. Since strains of LMP1 have been shown to differ in abundance between blood and throat washes, nasopharyngeal carcinomas (NPCs) from areas of endemicity and nonendemicity with matching blood were analyzed by using a heteroduplex tracking assay to distinguish LMP1 variants. Striking differences were found between the compartments with the Ch1 strain prevalent in the NPCs from areas of endemicity and nonendemicity and the B958 strain prevalent in the blood of the endemic samples, whereas multiple strains of LMP1 were prevalent in the blood of the nonendemic samples. The possible selection against the B958 strain appearing in the tumor was highly significant (P < 0.0001). Sequence analysis of the full-length LMP1 variants revealed changes in many of the known and computer-predicted HLA-restricted epitopes with changes in key positions in multiple, potential epitopes for the specific HLA of the patients. These amino acid substitutions at key positions in the LMP1 epitopes may result in a reduced cytotoxic-T-lymphocyte response. These data indicate that strains with specific variants of LMP1 are more likely to be found in NPC. The predominance of specific LMP1 variants in NPC could reflect differences in the biologic or molecular properties of the distinct forms of LMP1 or possible immune selection.

scholarly journals The GTPase Nog1 co-ordinates the assembly, maturation and quality control of distant ribosomal functional centers

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Purnima Klingauf-Nerurkar ◽  
Ludovic C Gillet ◽  
Daniela Portugal-Calisto ◽  
Michaela Oborská-Oplová ◽  
Martin Jäger ◽  
...  
Keyword(s):  
Quality Control ◽  
Protein Folding ◽  
Atpase Activity ◽  
Ribosomal Protein ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
Eukaryotic Ribosome ◽  
Ribosomal Stalk ◽  
Exit Tunnel ◽  
Ribosome Formation

Eukaryotic ribosome precursors acquire translation competence in the cytoplasm through stepwise release of bound assembly factors, and proofreading of their functional centers. In case of the pre-60S, these steps include removal of placeholders Rlp24, Arx1 and Mrt4 that prevent premature loading of the ribosomal protein eL24, the protein-folding machinery at the polypeptide exit tunnel (PET), and the ribosomal stalk, respectively. Here, we reveal that sequential ATPase and GTPase activities license release factors Rei1 and Yvh1 to trigger Arx1 and Mrt4 removal. Drg1-ATPase activity removes Rlp24 from the GTPase Nog1 on the pre-60S; consequently, the C-terminal tail of Nog1 is extracted from the PET. These events enable Rei1 to probe PET integrity and catalyze Arx1 release. Concomitantly, Nog1 eviction from the pre-60S permits peptidyl transferase center maturation, and allows Yvh1 to mediate Mrt4 release for stalk assembly. Thus, Nog1 co-ordinates the assembly, maturation and quality control of distant functional centers during ribosome formation.

scholarly journals Selection of sequence motifs and generative Hopfield-Potts models for protein families

2019 ◽  
Author(s):  
Kai Shimagaki ◽  
Martin Weigt
Keyword(s):  
Amino Acid ◽  
Ad Hoc ◽  
Three Dimensional ◽  
Generative Models ◽  
Amino Acid Sequences ◽  
Dimensional Structure ◽  
Sequence Motifs ◽  
Restricted Boltzmann Machines ◽  
Potts Models ◽  
Maximum Entropy Models

Statistical models for families of evolutionary related proteins have recently gained interest: in particular pairwise Potts models, as those inferred by the Direct-Coupling Analysis, have been able to extract information about the three-dimensional structure of folded proteins, and about the effect of amino-acid substitutions in proteins. These models are typically requested to reproduce the one- and two-point statistics of the amino-acid usage in a protein family, i.e. to capture the so-called residue conservation and covariation statistics of proteins of common evolutionary origin. Pairwise Potts models are the maximum-entropy models achieving this. While being successful, these models depend on huge numbers of ad hoc introduced parameters, which have to be estimated from finite amount of data and whose biophysical interpretation remains unclear. Here we propose an approach to parameter reduction, which is based on selecting collective sequence motifs. It naturally leads to the formulation of statistical sequence models in terms of Hopfield-Potts models. These models can be accurately inferred using a mapping to restricted Boltzmann machines and persistent contrastive divergence. We show that, when applied to protein data, even 20-40 patterns are sufficient to obtain statistically close-to-generative models. The Hopfield patterns form interpretable sequence motifs and may be used to clusterize amino-acid sequences into functional sub-families. However, the distributed collective nature of these motifs intrinsically limits the ability of Hopfield-Potts models in predicting contact maps, showing the necessity of developing models going beyond the Hopfield-Potts models discussed here.

scholarly journals Epitope Mapping of Antibodies against Prostate-specific Antigen with Use of Peptide Libraries

2002 ◽  
Vol 48 (12) ◽  
pp. 2208-2216 ◽  
Author(s):  
Jari Leinonen ◽  
Ping Wu ◽  
Ulf-Håkan Stenman
Keyword(s):  
Prostate Specific Antigen ◽  
Cyclic Peptides ◽  
Epitope Mapping ◽  
Specific Antigen ◽  
Peptide Libraries ◽  
Sequence Motifs ◽  
Large Panel ◽  
Distinct Sequence ◽  
Binding Peptides ◽  
Important Marker

Abstract Background: Prostate-specific antigen (PSA) is the most important marker for prostate cancer, but PSA concentrations determined by various assays can differ significantly because of differences in specificity of the antibodies used. To identify epitopes recognized by various monoclonal antibodies (MAbs) to PSA, we have isolated peptides that react with the paratopes of these. Methods: Six anti-PSA MAbs representing three major epitope groups were screened with five cyclic phage display peptide libraries. After selection, the peptide sequences were determined by sequencing of the relevant part of viral DNA. Binding of the phage peptides to the MAbs was monitored by immunoassay. Results: For each MAb, several paratope-binding peptides with distinct sequence motifs were identified, but only ∼10% showed similarity with the PSA sequence. Some of these correctly predicted the location of the epitopes. By sequential panning of the library with two closely related MAbs, we identified peptides reacting equally with both MAbs. When analyzed against a large panel of PSA MAbs, the peptides generally showed restricted specificity toward the MAb used for selection, but some peptides bound to several related MAbs. Conclusions: Most of the cyclic peptides selected with PSA MAbs are specific for the MAb used for selection and do not resemble any sequence on the antigen. Peptides reactive with two MAbs recognizing the same epitope can be obtained by sequential panning. This method can be used to predict the location of some epitopes, but additional methods are needed to confirm the result.

scholarly journals uS3/Rps3 controls fidelity of translation termination and programmed stop codon readthrough in co-operation with eIF3

2019 ◽  
Vol 47 (21) ◽  
pp. 11326-11343 ◽  
Author(s):  
Kristýna Poncová ◽  
Susan Wagner ◽  
Myrte Esmeralda Jansen ◽  
Petra Beznosková ◽  
Stanislava Gunišová ◽  
...  
Keyword(s):  
Translational Control ◽  
Stop Codon ◽  
Translation Termination ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation ◽  
C Terminus ◽  
Context Specific ◽  
Stop Codon Readthrough

Abstract Ribosome was long considered as a critical yet passive player in protein synthesis. Only recently the role of its basic components, ribosomal RNAs and proteins, in translational control has begun to emerge. Here we examined function of the small ribosomal protein uS3/Rps3, earlier shown to interact with eukaryotic translation initiation factor eIF3, in termination. We identified two residues in consecutive helices occurring in the mRNA entry pore, whose mutations to the opposite charge either reduced (K108E) or increased (R116D) stop codon readthrough. Whereas the latter increased overall levels of eIF3-containing terminating ribosomes in heavy polysomes in vivo indicating slower termination rates, the former specifically reduced eIF3 amounts in termination complexes. Combining these two mutations with the readthrough-reducing mutations at the extreme C-terminus of the a/Tif32 subunit of eIF3 either suppressed (R116D) or exacerbated (K108E) the readthrough phenotypes, and partially corrected or exacerbated the defects in the composition of termination complexes. In addition, we found that K108 affects efficiency of termination in the termination context-specific manner by promoting incorporation of readthrough-inducing tRNAs. Together with the multiple binding sites that we identified between these two proteins, we suggest that Rps3 and eIF3 closely co-operate to control translation termination and stop codon readthrough.

scholarly journals Structure of the murine rab3A gene: correlation of genomic organization with antibody epitopes

1993 ◽  
Vol 293 (1) ◽  
pp. 157-163 ◽  
Author(s):  
M Baumert ◽  
G Fischer von Mollard ◽  
R Jahn ◽  
T C Südhof
Keyword(s):  
Monoclonal Antibodies ◽  
Molecular Mass ◽  
Polyclonal Antibodies ◽  
Protein Domains ◽  
Amino Acid Sequences ◽  
Sequence Motifs ◽  
Rab Protein ◽  
Gtp Binding ◽  
Gene Structures ◽  
Antibody Epitopes

Rab3A is a neuronal low-molecular-mass GTP-binding protein that is modified post-translationally by two geranylgeranyl groups and specifically targeted to synaptic vesicles. We have now cloned and characterized the murine gene coding for rab3A. With a size of less than 8 kb including the promoter, the rab3A gene is relatively small. It contains five exons, the first of which is non-coding. The organization of the rab3A coding sequence into exons in the gene is different from that of ras proteins, the only other low-molecular-mass GTP-binding proteins with currently characterized gene structures. Nevertheless, the intron placement in the primary structure of rab3A may be indicative of a domain division of the protein, since each coding exon contains one of the four major conserved rab protein sequence motifs. The epitopes of monoclonal and polyclonal antibodies to rab3A were mapped with the hypothesis that antibody epitopes might represent distinct exposed protein domains and correlate with exon structures. Two monoclonal antibodies, named 42.1 and 42.2, were found to recognize epitopes with a different degree of conservation between different rab3 isoforms. These epitopes were mapped to relatively short amino acid sequences corresponding to exons 4 and 5 respectively, whereas a polyclonal antibody recognized a complex epitope that required the presence of intact rab3A. Comparison of the sequence of rab3A with that of ras, whose crystal structure has been determined, revealed that the epitopes for the monoclonal antibodies correspond to regions in ras that are highly exposed. Taken together, these results suggest that exons 4 and 5 at least represent distinct exposed protein domains that also form major natural epitopes in rab3A.

scholarly journals The nature of the purine at position 34 in tRNAs of 4-codon boxes is correlated with nucleotides at positions 32 and 38 to maintain decoding fidelity

2020 ◽  
Vol 48 (11) ◽  
pp. 6170-6183 ◽  
Author(s):  
Ketty Pernod ◽  
Laure Schaeffer ◽  
Johana Chicher ◽  
Eveline Hok ◽  
Christian Rick ◽  
...  
Keyword(s):  
Intergenic Region ◽  
Bacterial Genomes ◽  
Eukaryotic Translation ◽  
Eukaryotic Ribosome ◽  
Translation Fidelity ◽  
Translation Systems ◽  
Eukaryotic Genomes ◽  
Paralysis Virus ◽  
Decoding Fidelity

Abstract Translation fidelity relies essentially on the ability of ribosomes to accurately recognize triplet interactions between codons on mRNAs and anticodons of tRNAs. To determine the codon-anticodon pairs that are efficiently accepted by the eukaryotic ribosome, we took advantage of the IRES from the intergenic region (IGR) of the Cricket Paralysis Virus. It contains an essential pseudoknot PKI that structurally and functionally mimics a codon-anticodon helix. We screened the entire set of 4096 possible combinations using ultrahigh-throughput screenings combining coupled transcription/translation and droplet-based microfluidics. Only 97 combinations are efficiently accepted and accommodated for translocation and further elongation: 38 combinations involve cognate recognition with Watson-Crick pairs and 59 involve near-cognate recognition pairs with at least one mismatch. More than half of the near-cognate combinations (36/59) contain a G at the first position of the anticodon (numbered 34 of tRNA). G34-containing tRNAs decoding 4-codon boxes are almost absent from eukaryotic genomes in contrast to bacterial genomes. We reconstructed these missing tRNAs and could demonstrate that these tRNAs are toxic to cells due to their miscoding capacity in eukaryotic translation systems. We also show that the nature of the purine at position 34 is correlated with the nucleotides present at 32 and 38.

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