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 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 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 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 Structural and mutational analysis of the ribosome-arresting human XBP1u

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Vivekanandan Shanmuganathan ◽  
Nina Schiller ◽  
Anastasia Magoulopoulou ◽  
Jingdong Cheng ◽  
Katharina Braunger ◽  
...  
Keyword(s):  
Mutational Analysis ◽  
Central Component ◽  
Hydrophobic Region ◽  
Unfolded Protein ◽  
Translational Arrest ◽  
Exit Tunnel ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
Sec61 Channel ◽  
Mammalian Protein

XBP1u, a central component of the unfolded protein response (UPR), is a mammalian protein containing a functionally critical translational arrest peptide (AP). Here, we present a 3 Å cryo-EM structure of the stalled human XBP1u AP. It forms a unique turn in the ribosomal exit tunnel proximal to the peptidyl transferase center where it causes a subtle distortion, thereby explaining the temporary translational arrest induced by XBP1u. During ribosomal pausing the hydrophobic region 2 (HR2) of XBP1u is recognized by SRP, but fails to efficiently gate the Sec61 translocon. An exhaustive mutagenesis scan of the XBP1u AP revealed that only 8 out of 20 mutagenized positions are optimal; in the remaining 12 positions, we identify 55 different mutations increase the level of translational arrest. Thus, the wildtype XBP1u AP induces only an intermediate level of translational arrest, allowing efficient targeting by SRP without activating the Sec61 channel.

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 Increased freedom of movement in the nascent chain results in dynamic changes in the structure of the SecM arrest motif

2019 ◽  
Vol 39 (1) ◽  
Author(s):  
Hazel A. Bracken ◽  
Cheryl A. Woolhead
Keyword(s):  
Point Mutations ◽  
Translation Arrest ◽  
E Coli ◽  
Freedom Of Movement ◽  
Conserved Sequence ◽  
C Terminus ◽  
Nascent Chain ◽  
Translational Arrest ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel

Abstract Ribosomes are responsible for the synthesis of all cellular proteins. Due to the diversity of sequence and properties, it was initially believed that translating nascent chains would travel unhindered through the ribosome exit tunnel, however a small but increasing number of proteins have been identified that interact with the exit tunnel to induce translational arrest, Escherichia coli (E. coli) secretion monitor (SecM) is one such stalling peptide. How and why these peptides interact with the exit tunnel is not fully understood, however key features required for stalling appear to be an essential peptide arrest motif at the C-terminus and compaction of the nascent chain within the exit tunnel upon stalling. Mutagenesis of the SecM arrest sequence has identified three conservative point mutations that can retain a degree of stalling in this highly conserved sequence. This level of stalling is further increased when coupled with mutation of a non-essential arrest motif residue P153A. Further analysis of these mutants by pegylation assays indicates that this increase in stalling activity during translation is due to the ability of the P153A mutation to reintroduce compaction of the nascent chain within the exit tunnel possibly due to the improved flexibility of the nascent chain provided by the removal of a restrictive proline residue. The data presented here suggest that arrest sequences may be more prevalent and less highly conserved than previously thought, and highlight the significance of the interactions between the nascent chain and the exit tunnel to affecting translation arrest.

scholarly journals Structural and mutational analysis of the ribosome-arresting human XBP1u

2019 ◽  
Author(s):  
Vivekanandan Shanmuganathan ◽  
Nina Schiller ◽  
Anastasia Magoulopoulou ◽  
Jingdong Cheng ◽  
Katharina Braunger ◽  
...  
Keyword(s):  
Mutational Analysis ◽  
Central Component ◽  
Hydrophobic Region ◽  
Central Function ◽  
Unfolded Protein ◽  
Translational Arrest ◽  
Exit Tunnel ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
Mammalian Protein

AbstractXBP1u, a central component of the unfolded protein response (UPR), is a mammalian protein containing a functionally critical translational arrest peptide (AP). Here, we present a 3 Å cryo-EM structure of the stalled human XBP1u AP. It forms a unique turn in the upper part of the ribosomal exit tunnel and causes a subtle distortion of the peptidyl transferase center, explaining the temporary translational arrest induced by XBP1u. During ribosomal pausing the hydrophobic region 2 (HR2) of XBP1u is recognized by SRP, but fails to efficiently gate the Sec61 translocon. An exhaustive mutagenesis scan of the XBP1u AP revealed that only 10 out of 21 mutagenized positions in the XBP1u AP are optimal with respect to translational arrest activity. Thus, XBP1u has evolved to induce an intermediate level of translational arrest, allowing efficient targeting by SRP without activating the Sec61 channel and thereby serving its central function in the UPR.

scholarly journals Contributions of Molecular Dynamics Simulations to Elastohydrodynamic Lubrication

2021 ◽  
Vol 69 (1) ◽  
Author(s):  
James P. Ewen ◽  
Hugh A. Spikes ◽  
Daniele Dini
Keyword(s):  
Molecular Dynamics ◽  
Shear Rate ◽  
Experimental Design ◽  
Md Simulation ◽  
Elastohydrodynamic Lubrication ◽  
Quantitative Agreement ◽  
Coupling Methods ◽  
Fundamental Understanding ◽  
Shear Heating ◽  
Dynamics Simulations

AbstractThe prediction of friction under elastohydrodynamic lubrication (EHL) conditions remains one of the most important and controversial areas of tribology. This is mostly because the pressure and shear rate conditions inside EHL contacts are particularly severe, which complicates experimental design. Over the last decade, molecular dynamics (MD) simulation has played an increasingly significant role in our fundamental understanding of molecular behaviour under EHL conditions. In recent years, MD simulation has shown quantitative agreement with friction and viscosity results obtained experimentally, meaning that they can, either in isolation or through the use of multiscale coupling methods, begin to be used to test and inform macroscale models for EHL problems. This is particularly useful under conditions that are relevant inside machine components, but are difficult to obtain experimentally without uncontrollable shear heating.

scholarly journals Author response: The shape of the bacterial ribosome exit tunnel affects cotranslational protein folding

2018 ◽  
Author(s):  
Renuka Kudva ◽  
Pengfei Tian ◽  
Fátima Pardo-Avila ◽  
Marta Carroni ◽  
Robert B Best ◽  
...  
Keyword(s):  
Protein Folding ◽  
Author Response ◽  
Bacterial Ribosome ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel
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