Beckert, B. | bioRxiv Science

bioRxiv2024

Paenilamicins from the honey bee pathogen Paenibacillus larvae are context-specific translocation inhibitors of protein synthesis

The paenilamicins are a group of hybrid non-ribosomal peptide-polyketide compounds produced by the honey bee pathogen Paenibacillus larvae that display activity against Gram-positive pathogens, such as Staphylococcus aureus. While paenilamicins have been shown to inhibit protein synthesis, their mechanism of action has remained unclear. Here, we have determined structures of the paenilamicin PamB2 stalled ribosomes, revealing a unique binding site on the small 30S subunit located between the A- and P-site tRNAs. In addition to providing a precise description of interactions of PamB2 with the ribosome, the structures also rationalize the resistance mechanisms utilized by P. larvae. We could further demonstrate that PamB2 interferes with the translocation of mRNA and tRNAs through the ribosome during translation elongation, and that this inhibitory activity is influenced by the presence of modifications at position 37 of the A-site tRNA. Collectively, our study defines the paenilamicins as a new class of context-specific translocation inhibitors.

biochemistry↗

bioRxiv2024

A role for the S4-domain containing protein YlmH in ribosome-associated quality control in Bacillus subtilis

Ribosomes trapped on mRNAs during protein synthesis need to be rescued for the cell to survive. The most ubiquitous bacterial ribosome rescue pathway is trans-translation mediated by tmRNA and SmpB. Genetic inactivation of trans-translation can be lethal, unless the ribosomes are rescued by ArfA or ArfB alternative rescue factors or the ribosome-associated quality control (RQC) system, which in B. subtilis involves MutS2, RqcH, RqcP and Pth. Using transposon sequencing in a trans-translation-incompetent B. subtilis strain we identify a poorly characterized S4-domain-containing protein YlmH as a novel potential RQC factor. Cryo-EM structures reveal that YlmH binds peptidyl-tRNA-50S complexes in an position analogous to that of S4-domain-containing RqcP, and that, similarly to RqcP, YlmH can co-habit with RqcH. Consistently, we show that YlmH can assume the role of RqcP in RQC in facilitating the addition of polyalanine tails to the truncated nascent polypeptides. While in B. subtilis the function of YlmH is redundant with RqcP, our taxonomic analysis reveals that in multiple bacterial phyla RqcP is absent, while YlmH and RqcH are present, suggesting that in these species the YlmH plays a central role in the RQC.

microbiology↗

bioRxiv2022

Structural basis for translation inhibition 1 by the glycosylated antimicrobial peptide Drosocin from Drosophila melanogaster

The proline-rich antimicrobial peptide (PrAMP) drosocin is produced by Drosophila species to combat bacterial infection. Unlike many PrAMPs, drosocin is O-glycosylated at threonine 11, a post-translation modification that enhances its antimicrobial activity. Here we demonstrate that the O-glycosylation influences not only cellular uptake of the peptide, but also interacts with its intracellular target, the ribosome. Cryo-electron microscopy structures of glycosylated drosocin on the ribosome at 2.1-2.8 [A] resolution reveal that the peptide interferes with translation termination by binding within the polypeptide exit tunnel and trapping RF1 on the ribosome, reminiscent of that reported for the PrAMP apidaecin. The glycosylation of drosocin enables multiple interactions with U2609 of the 23S rRNA, leading to conformational changes that break the canonical base-pair with A752. Collectively, our study provides novel molecular insights into the interaction of O-glycosylated drosocin with the ribosome, which provides a structural basis for future development of this class of antimicrobials.

biochemistry↗

bioRxiv2022

DNA-measuring Wadjet SMC ATPases restrict smaller circular plasmids by DNA cleavage.

Structural maintenance of chromosomes (SMC) complexes fold DNA by loop extrusion to support chromosome segregation, genome maintenance, and gene expression. Wadjet systems (JetABCD/MksBEFG/EptABCD) are derivative SMC complexes with proposed roles in bacterial immunity against selfish DNA elements. Here, we show that JetABCD systems restrict extrachromosomal circular DNA with an upper size limit of about 100 kb, while a linear plasmid evades restriction. Recombinant preparations of a JetABCD complex cleave circular DNA regardless of its helical topology but not linear DNA; cleavage occurs at random positions and depends on ATP as well as the SMC ATPase. We solve a structure of the JetABCD core by cryo-EM revealing an alternative dimer-of-dimers configuration. The two SMC DNA motor units face in opposite orientations--rather than the same as observed with MukBEF--possibly representing a restriction-specific state of JetABCD. We hypothesize that JetABCD is a DNA-shape-specific endonuclease and present a model for the activation of DNA cleavage exclusively when extrusion of an entire plasmid has been completed by a single JetABCD complex. The self-stalling of two opposing DNA motor units may serve as signal to trigger DNA cleavage. Complete extrusion by a single complex cannot be achieved on the much larger chromosome, explaining how self-DNA may evade cleavage.

molecular biology↗

bioRxiv2022

Cryo-EM structures and binding of mouse ACE2 to SARS-CoV-2 variants of concern

Investigation of potential hosts of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is crucial to understanding future risks of spillover and spillback. SARS-CoV-2 has been reported to be transmitted from humans to various animals after requiring relatively few mutations.[1] There is significant interest in describing how the virus interacts with mice as they are well adapted to human environments, are used widely as infection models and can be infected.[2] Structural and binding data of the mouse ACE2 receptor with the Spike protein of newly identified SARS-CoV-2 variants are needed to better understand the impact of immune system evading mutations present in variants of concern (VOC). Previous studies have developed mouse-adapted variants and identified residues critical for binding to heterologous ACE2 receptors.[3,4] Here we report the cryo-EM structures of mouse ACE2 bound to trimeric Spike ectodomains of four different VOC: Beta, Omicron BA.1, Omicron BA.2.12.1 and Omicron BA.4/5. These variants represent the oldest to the newest variants known to bind the mouse ACE2 receptor. Our high-resolution structural data complemented with bio-layer interferometry (BLI) binding assays reveal a requirement for a combination of mutations in the Spike protein that enable binding to the mouse ACE2 receptor. AUTHOR SUMMARYThe SARS-CoV-2 virus can infect different types of animals beyond humans. The virus uses its Spike protein on its surface to bind to cells. These cells have a protein called ACE2 that the Spike protein recognizes. Animals have slightly different ACE2 receptors compared to humans. Mice are widely used as a research animal and live in the same environments as humans so scientists are particularly interested. Understanding how Spike proteins binds to the mouse ACE2 receptor allows us to understand the impact of immune evading mutations found in new variants. We use a high resolution imaging technique called cryo-electron microscopy to look at how different Spike variants bind to the ACE2 receptor from mouse at a resolution where we can see the amino acids. We can see directly the individual amino acids and mutations on the Spike protein that interact with the mouse ACE2 receptor. Many of the mutations found in variants of concern also increase the strength of binding to the mouse ACE2 receptor. This result suggests that mutations in the Spike protein of future variants may have an additional effect in influencing how it binds to not only human ACE2 receptors but to mice and also different animals.

immunology↗

bioRxiv2021

Structural analysis of the Spike of the Omicron SARS-COV-2 variant by Cryo-EM and implications for immune evasion

Investigation of potential hosts of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is crucial to understanding future risks of spillover and spillback. SARS-CoV-2 has been reported to be transmitted from humans to various animals after requiring relatively few mutations.[1] There is significant interest in describing how the virus interacts with mice as they are well adapted to human environments, are used widely as infection models and can be infected.[2] Structural and binding data of the mouse ACE2 receptor with the Spike protein of newly identified SARS-CoV-2 variants are needed to better understand the impact of immune system evading mutations present in variants of concern (VOC). Previous studies have developed mouse-adapted variants and identified residues critical for binding to heterologous ACE2 receptors.[3,4] Here we report the cryo-EM structures of mouse ACE2 bound to trimeric Spike ectodomains of four different VOC: Beta, Omicron BA.1, Omicron BA.2.12.1 and Omicron BA.4/5. These variants represent the oldest to the newest variants known to bind the mouse ACE2 receptor. Our high-resolution structural data complemented with bio-layer interferometry (BLI) binding assays reveal a requirement for a combination of mutations in the Spike protein that enable binding to the mouse ACE2 receptor. AUTHOR SUMMARYThe SARS-CoV-2 virus can infect different types of animals beyond humans. The virus uses its Spike protein on its surface to bind to cells. These cells have a protein called ACE2 that the Spike protein recognizes. Animals have slightly different ACE2 receptors compared to humans. Mice are widely used as a research animal and live in the same environments as humans so scientists are particularly interested. Understanding how Spike proteins binds to the mouse ACE2 receptor allows us to understand the impact of immune evading mutations found in new variants. We use a high resolution imaging technique called cryo-electron microscopy to look at how different Spike variants bind to the ACE2 receptor from mouse at a resolution where we can see the amino acids. We can see directly the individual amino acids and mutations on the Spike protein that interact with the mouse ACE2 receptor. Many of the mutations found in variants of concern also increase the strength of binding to the mouse ACE2 receptor. This result suggests that mutations in the Spike protein of future variants may have an additional effect in influencing how it binds to not only human ACE2 receptors but to mice and also different animals.

immunology↗

bioRxiv2020

Structure of Gcn1 bound to stalled and colliding 80S ribosomes

Protein synthesis is essential to cells and requires a constant supply of nutrients. Amino acid starvation leads to accumulation of uncharged tRNAs that promote ribosomal stalling, which is sensed by the protein kinase Gcn2, together with its effector proteins, Gcn1 and Gcn20. Activation of Gcn2 phosphorylates eIF2, leading to a global repression of translation. Fine-tuning of this adaptive response is performed by the Rbg2/Gir2 complex, which is a negative regulator of Gcn2. Despite the wealth of biochemical data, structures of Gcn proteins on the ribosome have remained elusive. Here we present a cryo-electron microscopy structure of the yeast Gcn1 protein in complex with stalled and colliding 80S ribosomes. Gcn1 interacts with both 80S ribosomes within the disome, such that the Gcn1 HEAT repeats span from the P-stalk region on the colliding ribosome to the A-site region of the lead ribosome. The lead ribosome is stalled in a non-rotated state with peptidyl-tRNA in the A-site, uncharged tRNA in the P-site, eIF5A in the E-site, as well as Rbg2/Gir2 located in the A-site factor binding region. By contrast, the colliding ribosome adopts a rotated state with peptidyl-tRNA in a hybrid A/P-site, uncharged-tRNA in the P/E-site and Mbf1 bound adjacent to the mRNA entry channel on the 40S subunit. Collectively, our findings provide a structural basis for Rbg2/Gir2 repression of Gcn2, and also reveal that colliding disomes are the substrate for Gcn1 binding, which has important implications not only for Gcn2-activated stress responses, but also for general ribosome quality control (RQC) pathways.

biochemistry↗

bioRxiv2020

eEF3 promotes late stages of tRNA translocation on the ribosome

SummaryIn addition to the conserved translation elongation factors eEF1A and eEF2, fungi require a third essential elongation factor, eEF3. While eEF3 has been implicated in tRNA binding and release at the A and E sites, its exact mechanism of action is unclear. Here we show that eEF3 acts at the mRNA–tRNA translocation step by promoting the dissociation of the tRNA from the E site, but independent of aminoacyl-tRNA recruitment to the A site. Depletion of eEF3 in vivo leads to a general slow-down in translation elongation due to accumulation of ribosomes with an occupied A site. Cryo-EM analysis of ex vivo eEF3-ribosome complexes shows that eEF3 facilitates late steps of translocation by favoring non-rotated ribosomal states as well as by opening the L1 stalk to release the E-site tRNA. Additionally, our analysis provides structural insights into novel translation elongation states, enabling presentation of a revised yeast translation elongation cycle.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗

bioRxiv2019

ResQ, a release factor-dependent ribosome rescue factor in the Gram-positive bacterium Bacillus subtilis

Rescue of the ribosomes from dead-end translation complexes, such as those on truncated (non-stop) mRNA, is essential for the cell. Whereas bacteria use trans-translation for ribosome rescue, some Gram-negative species possess alternative and release factor (RF)-dependent rescue factors, which enable an RF to catalyze stop codon-independent polypeptide release. We now discover that the Gram-positive Bacillus subtilis has an evolutionarily distinct ribosome rescue factor named ResQ. Genetic analysis shows that B. subtilis requires the function of either trans-translation or ResQ for growth, even in the absence of proteotoxic stresses. Biochemical and cryo-EM characterization demonstrates that ResQ binds to non-stop stalled ribosomes, recruits homologous RF2, but not RF1, and induces its transition into an open active conformation. Although ResQ is distinct from E. coli ArfA, they use convergent strategies in terms of mode of action and expression regulation, indicating that many bacteria may have evolved as yet unidentified ribosome rescue systems.

molecular biology↗

bioRxiv2019

The alarmone (p)ppGpp is part of the heat shock response of Bacillus subtilis

Here, B. subtilis was used as a model organism to investigate how cells respond and adapt to proteotoxic stress conditions. Our experiments suggested that the stringent response, caused by raised levels of the (p)ppGpp alarmone, plays a role during thermotolerance development and the heat shock response. Accordingly, our experiments revealed a rapid increase of cellular (p)ppGpp levels upon heat shock as well as salt- and oxidative stress. Strains lacking (p)ppGpp exhibited increased stress sensitivity, while raised (p)ppGpp levels conferred increased stress tolerance to heat- and oxidative stress. During thermotolerance development, stress response genes were highly up-regulated together with a concurrent transcriptional down-regulation of the rRNA, which was influenced by the second messenger (p)ppGpp and the transcription factor Spx. Remarkably, we observed that (p)ppGpp appeared to control the cellular translational capacity and that during heat stress the raised cellular levels of the alarmone were able to curb the rate of protein synthesis. Furthermore, (p)ppGpp controls the heat-induced expression of Hpf and thus the formation of translationally inactive 100S disomes. These results indicate that B. subtilis cells respond to heat-mediated protein unfolding and aggregation, not only by raising the cellular repair capacity, but also by decreasing translation involving (p)ppGpp mediated stringent response to concurrently reduce the protein load for the cellular protein quality control system.\n\nAuthor SummaryHere we demonstrate that the bacterial stringent response, which is known to slow down translation upon sensing nutrient starvation, is also intricately involved in the stress response of B. subtilis cells. The second messengers (p)ppGpp act as pleiotropic regulators during the adaptation to heat stress. (p)ppGpp slows down translation and is also involved in the transcriptional down-regulation of the translation machinery, together with the transcriptional stress regulator Spx. The stress-induced elevation of cellular (p)ppGpp levels confers increased stress tolerance and facilitates an improved protein homeostasis by reducing the load on the protein quality control system.

microbiology↗

bioRxiv2018

Structure of a hibernating 100S ribosome reveals an inactive conformation of the ribosomal protein S1

To survive under conditions of stress, such as nutrient deprivation, bacterial 70S ribosomes dimerize to form hibernating 100S particles1. In {gamma}-proteobacteria, such as Escherichia coli, 100S formation requires the ribosome modulation factor (RMF) and the hibernation promoting factor (HPF)2-4. Although structures of E. coli 100S particles have been reported5,6, the low resolution (18-38 [A]) prevented the mechanism of ribosome inactivation and dimerization to be fully elucidated. Here we present single particle cryo-electron microscopy structures of hibernating 70S and 100S particles isolated from stationary phase E. coli cells at 3.0-7.9 [A] resolution, respectively. Preferred orientation bias for the complete 100S particle was overcome using tilting during data collection. The structures reveal the binding sites for HPF and RMF as well as the unexpected presence of deacylated E-site tRNA and ribosomal protein S1 in the 100S particle. HPF interacts with the anticodon-stem-loop of the E-tRNA and occludes the binding site for the mRNA as well as A- and P-site tRNAs. RMF stabilizes a compact conformation of S1, which together sequester the anti-Shine-Dalgarno (SD) sequence of the 16S ribosomal RNA (rRNA), thereby inhibiting translation initiation. At the dimerization interface, S1 and S2 form intersubunit bridges with S3 and S4 and the C-terminus of S2 probes the mRNA entrance channel of the symmetry related particle, thus suggesting that only translationally inactive ribosomes are prone to dimerization. The back-to-back 100S dimerization mediated by HPF and RMF is distinct from that observed previously in Gram-positive bacteria7-10 and reveals a unique function for S1 in ribosome dimerization and inactivation, rather than its canonical role in facilitating translation initiation.

molecular biology↗