A point mutation in the nucleotide exchange factor eIF2B constitutively activates the integrated stress response by allosteric modulation

  1. Morgane Boone
  2. Lan Wang
  3. Rosalie Lawrence
  4. Adam Frost
  5. Peter Walter  Is a corresponding author
  6. Michael Schoof  Is a corresponding author
  1. Howard Hughes Medical Institute, University of California, San Francisco, United States
  2. 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.

The following data sets were generated

Article and author information

Author details

  1. Morgane Boone

    Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7807-5542
  2. Lan Wang

    Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8931-7201
  3. Rosalie Lawrence

    Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.
  4. Adam Frost

    Department of Biochemistry and Biophysics, University of California, San Francisco (Adjunct), San Francisco, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2231-2577
  5. Peter Walter

    Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States
    For correspondence
    peter@walterlab.ucsf.edu
    Competing interests
    Peter Walter, is an inventor on U.S. Patent 9708247 held by the Regents of the University of California that describes ISRIB and its analogs. Rights to the invention have been licensed by UCSF to Calico. For the remaining authors, no competing financial interests exist..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6849-708X
  6. Michael Schoof

    Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States
    For correspondence
    michael@walterlab.ucsf.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3531-5232

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.

Reviewing Editor

  1. Nahum Sonenberg, McGill University, Canada

Version history

  1. Preprint posted: December 6, 2021 (view preprint)
  2. Received: December 8, 2021
  3. Accepted: April 11, 2022
  4. Accepted Manuscript published: April 13, 2022 (version 1)
  5. Version of Record published: May 25, 2022 (version 2)
  6. Version of Record updated: May 31, 2022 (version 3)

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.

Metrics

  • 1,809
    views
  • 354
    downloads
  • 5
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Morgane Boone
  2. Lan Wang
  3. Rosalie Lawrence
  4. Adam Frost
  5. Peter Walter
  6. Michael Schoof
(2022)
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

Share this article

https://doi.org/10.7554/eLife.76171

Further reading

    1. Biochemistry and Chemical Biology
    Alexander S Baier, Nathan Gioacchini ... Craig L Peterson
    Research Article

    The yeast SWR1C chromatin remodeling enzyme catalyzes the ATP-dependent exchange of nucleosomal histone H2A for the histone variant H2A.Z, a key variant involved in a multitude of nuclear functions. How the 14-subunit SWR1C engages the nucleosomal substrate remains largely unknown. Studies on the ISWI, CHD1, and SWI/SNF families of chromatin remodeling enzymes have demonstrated key roles for the nucleosomal acidic patch for remodeling activity, however a role for this nucleosomal epitope in nucleosome editing by SWR1C has not been tested. Here, we employ a variety of biochemical assays to demonstrate an essential role for the acidic patch in the H2A.Z exchange reaction. Utilizing asymmetrically assembled nucleosomes, we demonstrate that the acidic patches on each face of the nucleosome are required for SWR1C-mediated dimer exchange, suggesting SWR1C engages the nucleosome in a ‘pincer-like’ conformation, engaging both patches simultaneously. Loss of a single acidic patch results in loss of high affinity nucleosome binding and nucleosomal stimulation of ATPase activity. We identify a conserved arginine-rich motif within the Swc5 subunit that binds the acidic patch and is key for dimer exchange activity. In addition, our cryoEM structure of a Swc5–nucleosome complex suggests that promoter proximal, histone H2B ubiquitylation may regulate H2A.Z deposition. Together these findings provide new insights into how SWR1C engages its nucleosomal substrate to promote efficient H2A.Z deposition.

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease
    Timothy A Bates, Mila Trank-Greene ... Fikadu G Tafesse
    Research Article

    Mycobacterium tuberculosis (Mtb) is known to survive within macrophages by compromising the integrity of the phagosomal compartment in which it resides. This activity primarily relies on the ESX-1 secretion system, predominantly involving the protein duo ESAT-6 and CFP-10. CFP-10 likely acts as a chaperone, while ESAT-6 likely disrupts phagosomal membrane stability via a largely unknown mechanism. we employ a series of biochemical analyses, protein modeling techniques, and a novel ESAT-6-specific nanobody to gain insight into the ESAT-6’s mode of action. First, we measure the binding kinetics of the tight 1:1 complex formed by ESAT-6 and CFP-10 at neutral pH. Subsequently, we demonstrate a rapid self-association of ESAT-6 into large complexes under acidic conditions, leading to the identification of a stable tetrameric ESAT-6 species. Using molecular dynamics simulations, we pinpoint the most probable interaction interface. Furthermore, we show that cytoplasmic expression of an anti-ESAT-6 nanobody blocks Mtb replication, thereby underlining the pivotal role of ESAT-6 in intracellular survival. Together, these data suggest that ESAT-6 acts by a pH-dependent mechanism to establish two-way communication between the cytoplasm and the Mtb-containing phagosome.