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 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 The shape of the bacterial ribosome exit tunnel affects cotranslational protein folding

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Renuka Kudva ◽  
Pengfei Tian ◽  
Fátima Pardo-Avila ◽  
Marta Carroni ◽  
Robert B Best ◽  
...  
Keyword(s):  
Protein Folding ◽  
Protein Domain ◽  
Coarse Grained ◽  
E Coli ◽  
Folded Structure ◽  
Bacterial Ribosome ◽  
Folded Proteins ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel ◽  
Dynamics Simulations

The E. coli ribosome exit tunnel can accommodate small folded proteins, while larger ones fold outside. It remains unclear, however, to what extent the geometry of the tunnel influences protein folding. Here, using E. coli ribosomes with deletions in loops in proteins uL23 and uL24 that protrude into the tunnel, we investigate how tunnel geometry determines where proteins of different sizes fold. We find that a 29-residue zinc-finger domain normally folding close to the uL23 loop folds deeper in the tunnel in uL23 Δloop ribosomes, while two ~ 100 residue proteins normally folding close to the uL24 loop near the tunnel exit port fold at deeper locations in uL24 Δloop ribosomes, in good agreement with results obtained by coarse-grained molecular dynamics simulations. This supports the idea that cotranslational folding commences once a protein domain reaches a location in the exit tunnel where there is sufficient space to house the folded structure.

scholarly journals Folding of VemP into translation-arresting secondary structure is driven by the ribosome exit tunnel

2021 ◽  
Author(s):  
Michal H Kolar ◽  
Gabor Nagy ◽  
John Kunkel ◽  
Sara M Vaiana ◽  
Lars V Bock ◽  
...  
Keyword(s):  
Small Molecules ◽  
Protein Production ◽  
Driving Forces ◽  
Peptidyl Transferase ◽  
Helix Formation ◽  
Translational Arrest ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel ◽  
Α Helix ◽  
Dynamics Simulations

The ribosome is a fundamental biomolecular complex responsible for protein production in cells. Nascent proteins emerge from the ribosome through a tunnel, where they may interact with the tunnel walls or small molecules such as antibiotics. These interactions can cause translational arrest with notable physiologic consequences. Here, we studied the arrest caused by the regulatory peptide VemP, which is known to form an α-helix in the ribosome tunnel near the peptidyl transferase center under specific conditions. We used all-atom molecular dynamics simulations of the entire ribosome and circular dichroism spectroscopy to study the driving forces of helix formation and how VemP causes the translational arrest. To that aim, we compared VemP dynamics in the ribosome tunnel with its dynamics in solution. We show that the VemP sequence has a low helical propensity in water and that the propensity is higher in more hydrophobic solvents. We propose that helix formation within the ribosome is driven by the tunnel environment and that a portion of VemP acts as an anchor. This anchor might slow down VemP progression through the tunnel enabling the α-helix formation, which causes the elongation arrest.

scholarly journals The Shape of the Bacterial Ribosome Exit Tunnel Affects Cotranslational Protein Folding

2018 ◽  
Author(s):  
Renuka Kudva ◽  
Pengfei Tian ◽  
Fatima Pardo Avila ◽  
Marta Carroni ◽  
Robert B. Best ◽  
...  
Keyword(s):  
Protein Folding ◽  
Protein Domain ◽  
Coarse Grained ◽  
E Coli ◽  
Folded Structure ◽  
Bacterial Ribosome ◽  
Folded Proteins ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel ◽  
Dynamics Simulations

AbstractThe E.coli ribosome exit tunnel can accommodate small folded proteins, while larger ones fold outside. It remains unclear, however, to what extent the geometry of the tunnel influences protein folding. Here, using E. coli ribosomes with deletions in loops in proteins uL23 and uL24 that protrude into the tunnel, we investigate how tunnel geometry determines where proteins of different sizes fold. We find that a 29-residue zinc-finger domain normally folding close to the uL23 loop folds deeper in the tunnel in uL23 Δloop ribosomes, while two ~100-residue protein normally folding close to the uL24 loop near the tunnel exit port fold at deeper locations in uL24 Δloop ribosomes, in good agreement with results obtained by coarse-grained molecular dynamics simulations. This supports the idea that cotranslational folding commences once a protein domain reaches a location in the exit tunnel where there is sufficient space to house the folded structure.

scholarly journals Structural basis for the tryptophan sensitivity of TnaC-mediated ribosome stalling

2021 ◽  
Author(s):  
Anne-Xander van der Stel ◽  
Emily R. Gordon ◽  
Arnab Sengupta ◽  
Allyson K. Martínez ◽  
Dorota Klepacki ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Structural Basis ◽  
Release Factor ◽  
Wild Type ◽  
Peptidyl Transferase ◽  
Ribosome Stalling ◽  
Bacterial Ribosome ◽  
Translational Arrest ◽  
Operon Expression ◽  
Recognition And Response

ABSTRACTFree L-tryptophan (L-Trp) induces the expression of the Escherichia coli tryptophanase operon, leading to the production of indole from L-Trp. Tryptophanase operon expression is controlled via a mechanism involving the tryptophan-dependent stalling of ribosomes engaged in translation of tnaC, a leader sequence upstream of tnaA that encodes a 24-residue peptide functioning as a sensor for L-Trp. Although extensive biochemical characterization has revealed the elements of the TnaC peptide and the ribosome that are responsible for translational arrest, the molecular mechanism underlying the recognition and response to L-Trp by the TnaC-ribosome complex remains unknown. Here, we use a combined biochemical and structural approach to characterize a variant of TnaC (R23F) in which stalling by L-Trp is enhanced because of reduced cleavage of TnaC(R23F)-peptidyl-tRNA. In contrast to previous data originated from lower resolution structural studies, we show that the TnaC–ribosome complex captures a single L-Trp molecule to undergo tryptophan-dependent termination arrest and that nascent TnaC prevents the catalytic GGQ loop of release factor 2 from adopting an active conformation at the peptidyl transferase center. In addition, we show that the conformation of the L-Trp binding site is not altered by the R23F mutation. This leads us to propose a model in which rates of TnaC-peptidyl-tRNA cleavage by release factor and binding of the L-Trp ligand to the translating ribosome determine the tryptophan sensitivity of the wild-type and mutant TnaC variants. Thus, our study reveals a strategy whereby a nascent peptide assists the bacterial ribosome in sensing a small metabolite.

scholarly journals Probing interplays between human XBP1u translational arrest peptide and 80S ribosome

2021 ◽  
Author(s):  
Francesco Di Palma ◽  
Sergio Decherchi ◽  
Fátima Pardo-Avila ◽  
Sauro Succi ◽  
Michael Levitt ◽  
...  
Keyword(s):  
Biological Process ◽  
Quantitative Agreement ◽  
Release Process ◽  
80S Ribosome ◽  
Clear Cut ◽  
Translational Arrest ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel ◽  
Dynamics Simulations ◽  
The Unfolded Protein Response

The ribosome stalling mechanism is a crucial biological process; yet its atomistic underpinning is still elusive. In this framework, the XBP1u translational arrest peptide (AP) plays a central role in regulating the Unfolded Protein Response (UPR) in eukaryotic cells. Here, we report multi-microseconds all atom molecular dynamics simulations designed to probe the interactions between the XBP1u AP and the mammalian ribosome exit tunnel, both for the wildtype AP and for four mutant variants of different arrest potency. Enhanced sampling simulations allow investigating the AP release process of the different variants shedding light on this complex mechanism. The present outcomes are in qualitative/quantitative agreement with available experimental data. In conclusion, we provide an unprecedented atomistic picture of this biological process and clear-cut insights into the key AP-ribosome interactions.

scholarly journals Common sequence motifs of nascent chains engage the ribosome surface and trigger factor

2021 ◽  
Vol 118 (52) ◽  
pp. e2103015118
Author(s):  
Annika Deckert ◽  
Anaïs M. E. Cassaignau ◽  
Xiaolin Wang ◽  
Tomasz Włodarski ◽  
Sammy H. S. Chan ◽  
...  
Keyword(s):  
Surface Interactions ◽  
Trigger Factor ◽  
Structural Basis ◽  
Sequence Motifs ◽  
Nascent Chain ◽  
Dynamics Simulations ◽  
Polypeptide Chains ◽  
Common Sequence

In the cell, the conformations of nascent polypeptide chains during translation are modulated by both the ribosome and its associated molecular chaperone, trigger factor. The specific interactions that underlie these modulations, however, are still not known in detail. Here, we combine protein engineering, in-cell and in vitro NMR spectroscopy, and molecular dynamics simulations to explore how proteins interact with the ribosome during their biosynthesis before folding occurs. Our observations of α-synuclein nascent chains in living Escherichia coli cells reveal that ribosome surface interactions dictate the dynamics of emerging disordered polypeptides in the crowded cytosol. We show that specific basic and aromatic motifs drive such interactions and directly compete with trigger factor binding while biasing the direction of the nascent chain during its exit out of the tunnel. These results reveal a structural basis for the functional role of the ribosome as a scaffold with holdase characteristics and explain how handover of the nascent chain to specific auxiliary proteins occurs among a host of other factors in the cytosol.

scholarly journals Analysis of the Genetic Variability of Virulence-Related Loci in Epidemic Clones of Methicillin-Resistant Staphylococcus aureus

2005 ◽  
Vol 49 (1) ◽  
pp. 366-379 ◽  
Author(s):  
A. R. Gomes ◽  
S. Vinga ◽  
M. Zavolan ◽  
H. de Lencastre
Keyword(s):  
Staphylococcus Aureus ◽  
Genetic Variability ◽  
Amino Acid Level ◽  
Point Of View ◽  
Sequence Motifs ◽  
Related Factors ◽  
Specific Sequence ◽  
Methicillin Resistant ◽  
Sequence Types ◽  
R Domain

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) isolates have previously been classified into major epidemic clonal types by pulsed-field gel electrophoresis in combination with multilocus sequence typing (MLST) and staphylococcal cassette chromosome mec typing. We aimed to investigate whether genetic variability in potentially polymorphic domains of virulence-related factors could provide another level of differentiation in a diverse collection of epidemic MRSA clones. The target regions of strains representative of epidemic clones and genetically related methicillin-susceptible S. aureus isolates from the 1960s that were sequenced included the R domains of clfA and clfB; the D, W, and M regions of fnbA and fnbB; and three regions in the agr operon. Sequence variation ranged from very conserved regions, such as those for RNAIII and the agr interpromoter region, to the highly polymorphic R regions of the clf genes. The sequences of the clf R domains could be grouped into six major sequence types on the basis of the sequences in their 3′ regions. Six sequence types were also observed for the fnb sequences at the amino acid level. From an evolutionary point of view, it was interesting that a small DNA stretch at the 3′ clf R-domain sequence and the fnb sequences agreed with the results of MLST for this set of strains. In particular, clfB R-domain sequences, which had a high discriminatory capacity and with which the types distinguished were congruent with those obtained by other molecular typing methods, have potential for use for the typing of S. aureus. Clone- and strain-specific sequence motifs in the clf and fnb genes may represent useful additions to a typing methodology with a DNA array.

Discovery of natural product ovalicin sensitive type 1 methionine aminopeptidases: molecular and structural basis

2019 ◽  
Vol 476 (6) ◽  
pp. 991-1003 ◽  
Author(s):  
Vijaykumar Pillalamarri ◽  
Tarun Arya ◽  
Neshatul Haque ◽  
Sandeep Chowdary Bala ◽  
Anil Kumar Marapaka ◽  
...  
Keyword(s):  
Natural Product ◽  
Active Site ◽  
Covalent Modification ◽  
Structural Basis ◽  
Wild Type ◽  
Methionine Aminopeptidases ◽  
Synthetic Derivative ◽  
Dynamics Simulations

Abstract Natural product ovalicin and its synthetic derivative TNP-470 have been extensively studied for their antiangiogenic property, and the later reached phase 3 clinical trials. They covalently modify the conserved histidine in Type 2 methionine aminopeptidases (MetAPs) at nanomolar concentrations. Even though a similar mechanism is possible in Type 1 human MetAP, it is inhibited only at millimolar concentration. In this study, we have discovered two Type 1 wild-type MetAPs (Streptococcus pneumoniae and Enterococcus faecalis) that are inhibited at low micromolar to nanomolar concentrations and established the molecular mechanism. F309 in the active site of Type 1 human MetAP (HsMetAP1b) seems to be the key to the resistance, while newly identified ovalicin sensitive Type 1 MetAPs have a methionine or isoleucine at this position. Type 2 human MetAP (HsMetAP2) also has isoleucine (I338) in the analogous position. Ovalicin inhibited F309M and F309I mutants of human MetAP1b at low micromolar concentration. Molecular dynamics simulations suggest that ovalicin is not stably placed in the active site of wild-type MetAP1b before the covalent modification. In the case of F309M mutant and human Type 2 MetAP, molecule spends more time in the active site providing time for covalent modification.

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