1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics

Structural and mutational analysis of the ribosome-arresting human XBP1u

  1. Vivekanandan Shanmuganathan
  2. Nina Schiller
  3. Anastasia Magoulopoulou
  4. Jingdong Cheng
  5. Katharina Braunger
  6. Florian Cymer
  7. Otto Berninghausen
  8. Birgitta Beatrix
  9. Kenji Kohno
  10. Gunnar von Heijne  Is a corresponding author
  11. Roland Beckmann  Is a corresponding author
  1. Ludwig Maximilian University of Munich, Germany
  2. Stockholm University, Sweden
  3. Nara Institute of Science and Technology, Japan
https://doi.org/10.7554/eLife.46267
Share this article
Cite this article
  1. Vivekanandan Shanmuganathan
  2. Nina Schiller
  3. Anastasia Magoulopoulou
  4. Jingdong Cheng
  5. Katharina Braunger
  6. Florian Cymer
  7. Otto Berninghausen
  8. Birgitta Beatrix
  9. Kenji Kohno
  10. Gunnar von Heijne
  11. Roland Beckmann
(2019)
Structural and mutational analysis of the ribosome-arresting human XBP1u
eLife 8:e46267.
https://doi.org/10.7554/eLife.46267
  2. Download BibTeX
  3. Download .RIS

Abstract

XBP1u, a central component of the unfolded protein response (UPR), is a mammalian protein containing a functionally critical translational arrest peptide (AP). Here, we present a 3 Å cryo-EM structure of the stalled human XBP1u AP. It forms a unique turn in the ribosomal exit tunnel proximal to the peptidyl transferase center where it causes a subtle distortion, thereby explaining the temporary translational arrest induced by XBP1u. During ribosomal pausing the hydrophobic region 2 (HR2) of XBP1u is recognized by SRP, but fails to efficiently gate the Sec61 translocon. An exhaustive mutagenesis scan of the XBP1u AP revealed that only 8 out of 20 mutagenized positions are optimal; in the remaining 12 positions, we identify 55 different mutations increase the level of translational arrest. Thus, the wildtype XBP1u AP induces only an intermediate level of translational arrest, allowing efficient targeting by SRP without activating the Sec61 channel.

Data availability

Generated models have been deposited in Protein Data Bank (PDB), and accessible via the following accession codes: 6R5Q, 6R6P, 6R6G and 6R7Q. Corresponding maps have been deposited in Electron Microscopy Data Bank (EMDB) and accessible with the following accession codes: EMDB-4729, EMDB-4737, EMDB-4735 and EMDB-4745.

The following data sets were generated

Article and author information

Author details

  1. Vivekanandan Shanmuganathan

    Gene Center, Department of Biochemistry, Ludwig Maximilian University of Munich, Munich, Germany
    Competing interests
    The authors declare that no competing interests exist.

  2. Nina Schiller

    Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden
    Competing interests
    The authors declare that no competing interests exist.

  3. Anastasia Magoulopoulou

    Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden
    Competing interests
    The authors declare that no competing interests exist.

  4. Jingdong Cheng

    Gene Center, Department of Biochemistry, Ludwig Maximilian University of Munich, Munich, Germany
    Competing interests
    The authors declare that no competing interests exist.

  5. Katharina Braunger

    Gene Center, Department of Biochemistry, Ludwig Maximilian University of Munich, Munich, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9067-2155

  6. Florian Cymer

    Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden
    Competing interests
    The authors declare that no competing interests exist.

  7. Otto Berninghausen

    Gene Center, Department of Biochemistry, Ludwig Maximilian University of Munich, Munich, Germany
    Competing interests
    The authors declare that no competing interests exist.

  8. Birgitta Beatrix

    Gene Center, Department of Biochemistry, Ludwig Maximilian University of Munich, Munich, Germany
    Competing interests
    The authors declare that no competing interests exist.

  9. Kenji Kohno

    Institute for Research Initiatives, Nara Institute of Science and Technology, Ikoma, Japan
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3503-6551

  10. Gunnar von Heijne

    Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden
    For correspondence
    gunnar.von.heijne@dbb.su.se
    Competing interests
    The authors declare that no competing interests exist.

  11. Roland Beckmann

    Gene Center, Department of Biochemistry, Ludwig Maximilian University of Munich, Munich, Germany
    For correspondence
    beckmann@genzentrum.lmu.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4291-3898

Funding

Deutsche Forschungsgemeinschaft (QBM (Quantitative Biosciences Munich) Graduate School Fellowship)

  • Vivekanandan Shanmuganathan

Knut och Alice Wallenbergs Stiftelse (2012.0282)

  • Gunnar von Heijne

Vetenskapsrådet (621-2014-3713)

  • Gunnar von Heijne

Cancerfonden (15 0888)

  • Gunnar von Heijne

Japan Society for the Promotion of Science (JP26116006)

  • Kenji Kohno

Deutsche Forschungsgemeinschaft (GRK1721)

  • Roland Beckmann

Boehringer Ingelheim Fonds (Graduate Fellowship)

  • Katharina Braunger

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Ramanujan S Hegde, MRC Laboratory of Molecular Biology, United Kingdom

Version history

  1. Received: February 20, 2019
  2. Accepted: June 20, 2019
  3. Accepted Manuscript published: June 27, 2019 (version 1)
  4. Version of Record published: July 11, 2019 (version 2)

Copyright

© 2019, Shanmuganathan 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

  • 4,802
    views
  • 648
    downloads
  • 58
    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. Vivekanandan Shanmuganathan
  2. Nina Schiller
  3. Anastasia Magoulopoulou
  4. Jingdong Cheng
  5. Katharina Braunger
  6. Florian Cymer
  7. Otto Berninghausen
  8. Birgitta Beatrix
  9. Kenji Kohno
  10. Gunnar von Heijne
  11. Roland Beckmann
(2019)
Structural and mutational analysis of the ribosome-arresting human XBP1u
eLife 8:e46267.
https://doi.org/10.7554/eLife.46267

Share this article

https://doi.org/10.7554/eLife.46267

Categories and tags

Further reading

    1. Biochemistry and Chemical Biology
    2. Neuroscience

    Alzheimer’s disease linked Aβ42 exerts product feedback inhibition on γ-secretase impairing downstream cell signaling

    Katarzyna Marta Zoltowska, Utpal Das ... Lucía Chávez-Gutiérrez
    Research Article

    Amyloid β (Aβ) peptides accumulating in the brain are proposed to trigger Alzheimer’s disease (AD). However, molecular cascades underlying their toxicity are poorly defined. Here, we explored a novel hypothesis for Aβ42 toxicity that arises from its proven affinity for γ-secretases. We hypothesized that the reported increases in Aβ42, particularly in the endolysosomal compartment, promote the establishment of a product feedback inhibitory mechanism on γ-secretases, and thereby impair downstream signaling events. We conducted kinetic analyses of γ-secretase activity in cell-free systems in the presence of Aβ, as well as cell-based and ex vivo assays in neuronal cell lines, neurons, and brain synaptosomes to assess the impact of Aβ on γ-secretases. We show that human Aβ42 peptides, but neither murine Aβ42 nor human Aβ17–42 (p3), inhibit γ-secretases and trigger accumulation of unprocessed substrates in neurons, including C-terminal fragments (CTFs) of APP, p75, and pan-cadherin. Moreover, Aβ42 treatment dysregulated cellular homeostasis, as shown by the induction of p75-dependent neuronal death in two distinct cellular systems. Our findings raise the possibility that pathological elevations in Aβ42 contribute to cellular toxicity via the γ-secretase inhibition, and provide a novel conceptual framework to address Aβ toxicity in the context of γ-secretase-dependent homeostatic signaling.

    1. Biochemistry and Chemical Biology
    2. Plant Biology

    Allosteric activation of the co-receptor BAK1 by the EFR receptor kinase initiates immune signaling

    Henning Mühlenbeck, Yuko Tsutsui ... Cyril Zipfel
    Research Article

    Transmembrane signaling by plant receptor kinases (RKs) has long been thought to involve reciprocal trans-phosphorylation of their intracellular kinase domains. The fact that many of these are pseudokinase domains, however, suggests that additional mechanisms must govern RK signaling activation. Non-catalytic signaling mechanisms of protein kinase domains have been described in metazoans, but information is scarce for plants. Recently, a non-catalytic function was reported for the leucine-rich repeat (LRR)-RK subfamily XIIa member EFR (elongation factor Tu receptor) and phosphorylation-dependent conformational changes were proposed to regulate signaling of RKs with non-RD kinase domains. Here, using EFR as a model, we describe a non-catalytic activation mechanism for LRR-RKs with non-RD kinase domains. EFR is an active kinase, but a kinase-dead variant retains the ability to enhance catalytic activity of its co-receptor kinase BAK1/SERK3 (brassinosteroid insensitive 1-associated kinase 1/somatic embryogenesis receptor kinase 3). Applying hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis and designing homology-based intragenic suppressor mutations, we provide evidence that the EFR kinase domain must adopt its active conformation in order to activate BAK1 allosterically, likely by supporting αC-helix positioning in BAK1. Our results suggest a conformational toggle model for signaling, in which BAK1 first phosphorylates EFR in the activation loop to stabilize its active conformation, allowing EFR in turn to allosterically activate BAK1.

    1. Biochemistry and Chemical Biology
    2. Cell Biology

    Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum

    Ya-Juan Wang, Xiao-Jing Di ... Ting-Wei Mu
    Research Article Updated

    Protein homeostasis (proteostasis) deficiency is an important contributing factor to neurological and metabolic diseases. However, how the proteostasis network orchestrates the folding and assembly of multi-subunit membrane proteins is poorly understood. Previous proteomics studies identified Hsp47 (Gene: SERPINH1), a heat shock protein in the endoplasmic reticulum lumen, as the most enriched interacting chaperone for gamma-aminobutyric acid type A (GABAA) receptors. Here, we show that Hsp47 enhances the functional surface expression of GABAA receptors in rat neurons and human HEK293T cells. Furthermore, molecular mechanism study demonstrates that Hsp47 acts after BiP (Gene: HSPA5) and preferentially binds the folded conformation of GABAA receptors without inducing the unfolded protein response in HEK293T cells. Therefore, Hsp47 promotes the subunit-subunit interaction, the receptor assembly process, and the anterograde trafficking of GABAA receptors. Overexpressing Hsp47 is sufficient to correct the surface expression and function of epilepsy-associated GABAA receptor variants in HEK293T cells. Hsp47 also promotes the surface trafficking of other Cys-loop receptors, including nicotinic acetylcholine receptors and serotonin type 3 receptors in HEK293T cells. Therefore, in addition to its known function as a collagen chaperone, this work establishes that Hsp47 plays a critical and general role in the maturation of multi-subunit Cys-loop neuroreceptors.