Structural and mutational analysis of the ribosome-arresting human XBP1u

Shanmuganathan, Vivekanandan; Schiller, Nina; Magoulopoulou, Anastasia; Cheng, Jingdong; Braunger, Katharina; Cymer, Florian; Berninghausen, Otto; Beatrix, Birgitta; Kohno, Kenji; Heijne, Gunnar von; Beckmann, Roland

doi:10.7554/elife.46267

Cited by 66 publications

(76 citation statements)

References 63 publications

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“…(F and G) Comparison of RqcH and tRNA within (F) State A and (G) State B. (KH and I) Comparison of tRNAs (cyan) from (H) State A or (I) State B with classical A-and P-site tRNAs (PDB 6CFJ)(Tereshchenkov et al, 2018) or hybrid A/P-site tRNA (PDB 6R6P)(Shanmuganathan et al, 2019). (J) Superposition of RqcH from states A (pink) and B (purple) with degrees of movement indicated.…”

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confidence: 99%

Structural basis for bacterial ribosome quality control

Crowe‐McAuliffe

Takada

Murina

et al. 2020

Preprint

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SummaryIn all branches of life, stalled translation intermediates are recognized and processed by ribosome-associated quality-control (RQC) pathways. RQC begins with splitting of stalled ribosomes, leaving an unfinished polypeptide still attached to the large subunit. Ancient and conserved NEMF family RQC proteins target these incomplete proteins for degradation by the addition of C-terminal ‘tails.’ How such tailing can occur without the regular suite of translational components is, however, unclear. Using ex vivo single-particle cryo-EM, we show that C-terminal tailing in Bacillus subtilis is mediated by NEMF protein RqcH in concert with YabO, a protein homologous to, yet distinct from, Hsp15. Our structures reveal how these factors mediate tRNA movement across the ribosomal 50S subunit to synthesize polypeptides in the absence of mRNA or the small subunit.

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confidence: 99%

Structural basis for bacterial ribosome quality control

Crowe‐McAuliffe

Takada

Murina

et al. 2020

Preprint

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“…4 and Fig. 5) 10, [38][39][40][41] . For example, the hCMV stalling peptide forms an α-helix in the exit tunnel, but translation termination is mainly inhibited by two C-terminal prolines (Fig.…”

Section: Discussionmentioning

confidence: 99%

Selective inhibition of human translation by a drug-like compound that traps terminated protein nascent chains on the ribosome

Fr³

et al. 2020

Preprint

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Methods to directly inhibit gene expression using small molecules hold promise for the development of new therapeutics targeting proteins that have evaded previous attempts at drug discovery. Among these, small molecules including the drug-like compound PF-06446846 (PF846) selectively inhibit the synthesis of specific proteins, by stalling translation elongation 1-4 . These molecules also inhibit translation termination 4 by an unknown mechanism. Using cryo-electron microscopy (cryo-EM) and biochemical approaches, we show that PF846 arrests translation at the stop codon by slowing hydrolysis of the protein nascent chain (NC) from peptidyl-site (P-site) tRNA by eukaryotic release factor 1 (eRF1). After NC hydrolysis from the P-site tRNA, PF846 traps the NC in the ribosome exit tunnel in a compact α-helical conformation that induces 28S rRNA nucleotide rearrangements propagating back to the ribosome peptidyl transferase center (PTC). Mutational analyses and human cell-based experiments elucidate the pivotal amino acids of the NC required for PF846-dependent termination arrest, all of which face the PF846 side of the ribosome exit tunnel. The structural and functional data support a model in which PF846 inhibits translation termination by inducing allosteric conformational rearrangements in the NC and PTC that suppress peptidyl-tRNA hydrolysis promoted by eRF1, and trap the NC in the ribosome exit tunnel. This unprecedented mechanism of action reveals new principles of translation termination and lays the foundation for new therapeutic strategies.Li et al.3 MainMany diseases are affected by proteins which have been difficult or impossible to target directly with small molecule therapeutics. To treat these diseases, efforts to expand the druggable proteome have attempted to harness knowledge of endogenous cellular pathways to alter gene expression. For example, compounds termed PROteolysis TArgeting Chimeras (PROTACs) serve as molecular recruiters that can direct specific proteins to the ubiquitin-proteasome protein degradation machinery 5 .Other strategies use small molecules that bind messenger RNAs (mRNAs) to inhibit their translation, or lead to RNA degradation by cellular quality control systems 6 .Recently, small molecules have even been developed to direct secreted proteins to the lysosome for degradation 7 . However, ensuring the selectivity of these new small molecule-based strategies is complicated by the fact that they tap into endogenous regulatory pathways with a large number of physiological substrates, requiring a deep understanding of the molecular basis for targeting specificity.Recently we described the drug-like compound PF846 and its derivatives that target proteins by inhibiting the translation of specific mRNAs by the human ribosome 1-4 . These small molecules bind in the ribosome exit tunnel in a eukaryotic-specific pocket

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“…The 3.0-Å cryo-EM structure of XBP1u-paused Oryctolagus cuniculus ribosome-nascent chain complex (PDB: 6R5Q (Shanmuganathan et al, 2019)) was used as a starting model for structure refinement. The 60S subunit was refined into the 3.1 Å cryo-EM map with PR 20 , which was remodeled in Coot, starting with the best fitting N-out conformation from the yeast 80S-PR 20 complex.…”

Section: Rabbit 80s Complexmentioning

confidence: 99%

Ribosome inhibition byC9ORF72-ALS/FTD-associated poly-PR and poly-GR proteins revealed by cryo-EM

Loveland

Svidritskiy

Susorov

et al. 2020

Preprint

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Toxic dipeptide repeat (DPR) proteins are produced from expanded G4C2 hexanucleotide repeats in the C9ORF72 gene, which cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Two DPR proteins, poly-PR and poly-GR, repress cellular translation but the molecular mechanism remains unknown. Here we show that poly-PR and poly-GR of ≥ 20 repeats inhibit the ribosome’s peptidyl-transferase activity at nanomolar concentrations, comparable to specific translation inhibitors. High-resolution cryo-EM structures reveal that poly-PR and poly-GR block the polypeptide tunnel of the ribosome, extending into the peptidyl-transferase center. Consistent with these findings, the macrolide erythromycin, which binds in the tunnel, competes with the DPR proteins and restores peptidyl-transferase activity. Our results demonstrate that strong and specific binding of poly-PR and poly-GR in the ribosomal tunnel blocks translation, revealing the structural basis of their toxicity in C9ORF72-ALS/FTD.

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Structural and mutational analysis of the ribosome-arresting human XBP1u

Cited by 66 publications

References 63 publications

Structural basis for bacterial ribosome quality control

Structural basis for bacterial ribosome quality control

Selective inhibition of human translation by a drug-like compound that traps terminated protein nascent chains on the ribosome

Ribosome inhibition byC9ORF72-ALS/FTD-associated poly-PR and poly-GR proteins revealed by cryo-EM

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