Biology subjects

Leibundgut, M.

Publications and source records attributed to Leibundgut, M..

10 recordsLinked to original sources

bioRxiv2026

C-terminal evolutionary remodelling of isoleucyl-tRNA synthetases is a prokaryote-specific strategy for tuning aminoacylation rate

Aminoacyl-tRNA synthetases are the guardians of translational fidelity. Their complex function is mirrored by an elaborate structure, which includes multiple nested domains. While the evolutionary pressures that promoted the emergence of some domains, such as the editing domain, are clear, the pressures acting on other domains, particularly those at the C-terminus, are not. Here, we use a combination of kinetic analysis, X-ray crystallography, and bioinformatics to unveil the history and evolutionary forces that have shaped isoleucyl-tRNA synthetase (IleRS) domain structure. We find that the traditional classification into IleRS1 and IleRS2, based on the C-terminal tRNA-recognition domains, is incomplete, as it fails to capture features of the synthetic domain. Guided by the crystal structure of the Priestia megaterium IleRS2:tRNA complex, we removed key interactions between IleRS2 and its cognate tRNA and characterised their impact on enzyme activity. We found that D-loop interactions with the IleRS2 C-terminal region are non-essential in prokaryotes, and their loss can even increase catalytic turnover. Further, the zinc-binding domain of IleRS1 recognises the anticodon less stringently than the canonical C-terminal domain of IleRS2. Our data suggest that C-terminal evolutionary remodelling of IleRSs is an ongoing process with a historical precedent, consistent with selection for faster aminoacylation rate.

biochemistry↗

bioRxiv2023

Universal features of Nsp1-mediated translational shutdown by coronaviruses

Nonstructural protein 1 (Nsp1) produced by coronaviruses shuts down host protein synthesis in infected cells. The C-terminal domain of SARS-CoV-2 Nsp1 was shown to bind to the small ribosomal subunit to inhibit translation, but it is not clear whether this mechanism is broadly used by coronaviruses, whether the N-terminal domain of Nsp1 binds the ribosome, or how Nsp1 specifically permits translation of viral mRNAs. Here, we investigated Nsp1 from three representative Betacoronaviruses - SARS-CoV-2, MERS-CoV, and Bat-Hp-CoV - using structural, biophysical, and biochemical assays. We revealed a conserved mechanism of host translational shutdown across the three coronaviruses. We further demonstrated that the N-terminal domain of Bat-Hp-CoV Nsp1 binds to the decoding center of the 40S subunit, where it would prevent mRNA and eIF1A binding. Structure-based biochemical experiments identified a conserved role of these inhibitory interactions in all three coronaviruses and showed that the same regions of Nsp1 are responsible for the preferential translation of viral mRNAs. Our results provide a mechanistic framework to understand how Betacoronaviruses overcome translational inhibition to produce viral proteins.

biochemistry↗

bioRxiv2023

Antibiotic hyper-resistance in a class I aminoacyl-tRNA synthetase with altered active site signature motif

Antibiotics target key biological processes that include protein synthesis. Bacteria respond by developing resistance, which increases rapidly due to antibiotics overuse. Mupirocin, a clinically used natural antibiotic, inhibits isoleucyl-tRNA synthetase (IleRS), an enzyme that links isoleucine to its tRNAIle for protein synthesis. Two IleRSs, mupirocin-sensitive IleRS1 and resistant IleRS2, coexist in bacteria. The latter may also be found in resistant Staphylococcus aureus clinical isolates. Here, we describe the structural basis of mupirocin resistance and unravel a mechanism of hyper-resistance evolved by some IleRS2 proteins. We surprisingly find that an up to 103-fold increase in resistance originates from alteration of the HIGH motif, a signature motif of the class I aminoacyl-tRNA synthetases to which IleRSs belong. The structural analysis demonstrates how an altered HIGH motif could be adopted in IleRS2 but not IleRS1, providing insight into an elegant mechanism for coevolution of the key catalytic motif and associated antibiotic resistance.

biochemistry↗

bioRxiv2022

Molecular basis of the TRAP complex function in ER protein biogenesis

The Translocon Associated Protein (TRAP) complex resides in the endoplasmic reticulum (ER) membrane and interacts with the Sec translocon and the translating ribosome to facilitate biogenesis of secretory and membrane proteins1-4. TRAP is essential for the secretion of many hormones, and its key role in the production of the hormone peptide insulin has been particularly well established5,6. The mechanism by which TRAP engages ribosomes and the translocon to facilitate translocation of protein clients in the secretory pathway is not clear. Here, we reveal the molecular architecture of the mammalian TRAP complex and how it engages the translating ribosome associated with Sec61 translocon on the ER membrane. The TRAP complex is anchored to the ribosome via a long tether and its position relative to the ribosome and the translocon is further stabilized by a finger-like loop. This spatial arrangement positions a cradle-like lumenal domain of TRAP below the protein conducting pore of the translocon for interactions with translocated nascent chains. The biological importance of these key interactions is evident by structure-guided TRAP mutations in C. elegans that lead to growth deficits associated with increased ER stress and defects in insulin secretion. Our findings elucidate the molecular basis of the TRAP complex in the biogenesis and translocation of proteins at the ER.

molecular biology↗

bioRxiv2021

Stepwise maturation of the peptidyl transferase region of human mitoribosomes

Mitochondrial ribosomes are specialized for the synthesis of membrane proteins responsible for oxidative phosphorylation. Mammalian mitoribosomes diverged considerably from the ancestral bacterial ribosomes and feature dramatically reduced ribosomal RNAs. Structural basis of the mammalian mitochondrial ribosome assembly is currently not understood. Here we present eight distinct assembly intermediates of the human large mitoribosomal subunit involving 7 assembly factors. We discover that NSUN4-MTERF4 dimer plays a critical role in the process by stabilizing the 16S rRNA in a conformation that exposes the functionally important regions of rRNA for modification by MRM2 methyltransferase and quality control interactions with a conserved mitochondrial GTPase MTG2 that contacts the sarcin ricin loop and the immature active site. The successive action of these factors leads to the formation of the peptidyl transferase active site of the mitoribosome and the folding of the surrounding rRNA regions responsible for interactions with tRNAs and the small ribosomal subunit.

biochemistry↗

bioRxiv2020

Evolution of a virus-like architecture and packaging mechanism in arepurposed bacterial protein

Viruses are ubiquitous pathogens of global impact. Prompted by the hypothesis that their earliest progenitors recruited host proteins for virion formation, we have used stringent laboratory evolution to convert a bacterial enzyme lacking affinity for nucleic acids into an artificial nucleocapsid that efficiently packages and protects multiple copies of its own encoding mRNA. Revealing remarkable convergence on the molecular hallmarks of natural viruses, the accompanying changes reorganized the protein building blocks into an interlaced 240-subunit icosahedral capsid impermeable to nucleases, while emergence of a robust RNA stem-loop packaging cassette ensured high encapsidation yields and specificity. In addition to evincing a plausible evolutionary pathway for primordial viruses, these findings highlight practical strategies for developing non-viral carriers for diverse vaccine and delivery applications.

biochemistry↗

bioRxiv2020

Structural basis of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome

Programmed ribosomal frameshifting is the key event during translation of the SARS-CoV-2 RNA genome allowing synthesis of the viral RNA-dependent RNA polymerase and downstream viral proteins. Here we present the cryo-EM structure of the mammalian ribosome in the process of translating viral RNA paused in a conformation primed for frameshifting. We observe that the viral RNA adopts a pseudoknot structure lodged at the mRNA entry channel of the ribosome to generate tension in the mRNA that leads to frameshifting. The nascent viral polyprotein that is being synthesized by the ribosome paused at the frameshifting site forms distinct interactions with the ribosomal polypeptide exit tunnel. We use biochemical experiments to validate our structural observations and to reveal mechanistic and regulatory features that influence the frameshifting efficiency. Finally, a compound previously shown to reduce frameshifting is able to inhibit SARS-CoV-2 replication in infected cells, establishing coronavirus frameshifting as target for antiviral intervention.

molecular biology↗

bioRxiv2020

SARS-CoV-2 Nsp1 binds ribosomal mRNA channel to inhibit translation

The non-structural protein 1 (Nsp1), also referred to as the host shutoff factor, is the first viral protein that is synthesized in SARS-CoV-2 infected human cells to suppress host innate immune functions1,2. By combining cryo-electron microscopy and biochemical experiments, we show that SARS-CoV-2 Nsp1 binds to the human 40S subunit in ribosomal complexes including the 43S pre-initiation complex. The protein inserts its C-terminal domain at the entrance to the mRNA channel where it interferes with mRNA binding. We observe potent translation inhibition in the presence of Nsp1 in lysates from human cells. Based on the high-resolution structure of the 40S-Nsp1 complex, we identify residues of Nsp1 crucial for mediating translation inhibition. We further show that the full-length 5’ untranslated region of the genomic viral mRNA stimulates translation in vitro, suggesting that SARS-CoV-2 combines inhibition of translation by Nsp1 with efficient translation of the viral mRNA to achieve expression of viral genes3.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗

bioRxiv2019

Structure and functional implications of WYL-domain-containing transcription factor PafBC involved in the mycobacterial DNA damage response

In mycobacteria, transcriptional activator PafBC is responsible for upregulating the majority of genes induced by DNA damage. Understanding the mechanism of PafBC activation is impeded by a lack of structural information on this transcription factor that contains a widespread, but poorly understood WYL domain frequently encountered in bacterial transcription factors. Here, we determined the crystal structure of Arthrobacter aurescens PafBC. The protein consists of two modules, each harboring an N-terminal helix-turn-helix DNA binding domain followed by a central WYL and a C-terminal extension (WCX) domain. The WYL domains exhibit Sm-folds, while the WCX domains adopt ferredoxin-like folds, both characteristic for RNA binding proteins. Our results suggest a mechanism of regulation in which WYL domain-containing transcription factors may be activated by binding RNA molecules. Using an in vivo mutational screen in Mycobacterium smegmatis, we identify potential co-activator binding sites on PafBC.

molecular biology↗

bioRxiv2017

Structure of the eukaryotic cytoplasmic pre-40S ribosomal subunit

Final maturation of eukaryotic ribosomes occurs in the cytoplasm and requires the sequential removal of associated assembly factors and processing of the immature 20S pre-RNA. Using cryo-electron microscopy (cryo-EM), we have determined the structure of a cytoplasmic pre-40S particle poised to initiate final maturation at a resolution of 3.4 [A]. The structure reveals the extent of conformational rearrangements of the 3 major and 3 minor domains of the ribosomal RNA that take place during maturation, as well as the roles of the assembly factors Enp1, Ltv1, Rio2, Tsr1, and Pno1 in the process. Altogether, we provide a structural framework for the coordination of the final maturation events that drive a pre-40S particle towards the mature form capable of engaging in translation.

molecular biology↗