A point mutation in the nucleotide exchange factor eIF2B constitutively activates the integrated stress response by allosteric modulation
- Howard Hughes Medical Institute, University of California, San Francisco, United States
- University of California, San Francisco (Adjunct), United States
Abstract
In eukaryotic cells, stressors reprogram the cellular proteome by activating the integrated stress response (ISR). In its canonical form, stress-sensing kinases phosphorylate the eukaryotic translation initiation factor eIF2 (eIF2-P), which ultimately leads to reduced levels of ternary complex required for initiation of mRNA translation. Previously we showed that translational control is primarily exerted through a conformational switch in eIF2's nucleotide exchange factor, eIF2B, which shifts from its active A-State conformation to its inhibited I-State conformation upon eIF2-P binding, resulting in reduced nucleotide exchange on eIF2 (Schoof et al. 2021). Here, we show functionally and structurally how a single histidine to aspartate point mutation in eIF2B's β subunit (H160D) mimics the effects of eIF2-P binding by promoting an I-State like conformation, resulting in eIF2-P independent activation of the ISR. These findings corroborate our previously proposed A/I-State model of allosteric ISR regulation.
Data availability
All data generated or anaysed during this study are included in the manuscript and source data files. The final structural model has been deposited in PDB under the accession code 7TRJ. Amplicon sequencing data for the CRISPR clones has been deposited in NCBI's Sequence Read Archive (SRA) under accession number PRJNA821864.
Article and author information
Author details
Funding
Calico Life Sciences LLC
- Peter Walter
The George and Judy Marcus Family Foundation
- Peter Walter
Belgian-American Educational Foundation
- Morgane Boone
Damon-Runyon Cancer Research Foundation
- Lan Wang
Jan Coffin Child Foundation
- Rosalie Lawrence
Chan Zuckerberg Biohub Investigator Award
- Adam Frost
HHMI Faculty Scholar grant
- Adam Frost
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2022, Boone et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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A point mutation in the nucleotide exchange factor eIF2B constitutively activates the integrated stress response by allosteric modulation
eLife 11:e76171.
https://doi.org/10.7554/eLife.76171
Further reading
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Teichoic acids (TA) are linear phospho-saccharidic polymers and important constituents of the cell envelope of Gram-positive bacteria, either bound to the peptidoglycan as wall teichoic acids (WTA) or to the membrane as lipoteichoic acids (LTA). The composition of TA varies greatly but the presence of both WTA and LTA is highly conserved, hinting at an underlying fundamental function that is distinct from their specific roles in diverse organisms. We report the observation of a periplasmic space in Streptococcus pneumoniae by cryo-electron microscopy of vitreous sections. The thickness and appearance of this region change upon deletion of genes involved in the attachment of TA, supporting their role in the maintenance of a periplasmic space in Gram-positive bacteria as a possible universal function. Consequences of these mutations were further examined by super-resolved microscopy, following metabolic labeling and fluorophore coupling by click chemistry. This novel labeling method also enabled in-gel analysis of cell fractions. With this approach, we were able to titrate the actual amount of TA per cell and to determine the ratio of WTA to LTA. In addition, we followed the change of TA length during growth phases, and discovered that a mutant devoid of LTA accumulates the membrane-bound polymerized TA precursor.
