Auto-regulation of Rab5 GEF activity in Rabex5 by allosteric structural changes, catalytic core dynamics and ubiquitin binding

  1. Janelle Lauer
  2. Sandra Segeletz
  3. Alice Cezanne
  4. Giambattista Guaitoli
  5. Francesco Raimondi
  6. Marc Gentzel
  7. Vikram Alva
  8. Michael Habeck
  9. Yannis Kalaidzidis
  10. Marius Ueffing
  11. Andrei N Lupas
  12. Christian Johannes Gloeckner
  13. Marino Zerial  Is a corresponding author
  1. Max Planck Institute of Molecular Cell Biology and Genetics, Germany
  2. German Center for Neurodegenerative Diseases, Germany
  3. Heidelberg University, Germany
  4. Technical University Dresden, Germany
  5. Max Planck Institute for Developmental Biology, Germany
  6. Max Planck Institute for Biophysical Chemistry, Germany
  7. University of Tübingen, Germany

Abstract

Intracellular trafficking depends on the function of Rab GTPases, whose activation is regulated by guanine exchange factors (GEFs). The Rab5 GEF, Rabex5, was previously proposed to be auto-inhibited by its C-terminus. Here, we studied full-length Rabex5 and Rabaptin5 proteins as well as domain deletion Rabex5 mutants using hydrogen deuterium exchange mass spectrometry. We generated a structural model of Rabex5, using chemical cross-linking mass spectrometry and integrative modeling techniques. By correlating structural changes with nucleotide exchange activity for each construct, we uncovered new auto-regulatory roles for the Ubiquitin binding domains and the Linker connecting those domains to the catalytic core of Rabex5. We further provide evidence that enhanced dynamics in the catalytic core are linked to catalysis. Our results suggest a more complex auto-regulation mechanism than previously thought and imply that Ubiquitin binding serves not only to position Rabex5 but to also control its Rab5 GEF activity through allosteric structural alterations.

Data availability

Data generated for figures 1a and 3 are included in the supporting files.

Article and author information

Author details

  1. Janelle Lauer

    Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1412-6766
  2. Sandra Segeletz

    Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
    Competing interests
    The authors declare that no competing interests exist.
  3. Alice Cezanne

    Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
    Competing interests
    The authors declare that no competing interests exist.
  4. Giambattista Guaitoli

    Translational Biomarker in Neurodegenerative Diseases, German Center for Neurodegenerative Diseases, Tübingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  5. Francesco Raimondi

    Bioquant, Heidelberg University, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  6. Marc Gentzel

    Molecular Analysis Mass Spectrometry Center for Molecular and Cellular Bioimaging, Technical University Dresden, Dresden, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4482-6010
  7. Vikram Alva

    Department of Protein Evolution, Max Planck Institute for Developmental Biology, Tübingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1188-473X
  8. Michael Habeck

    Statistical Inverse Problems in Biophysics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  9. Yannis Kalaidzidis

    Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
    Competing interests
    The authors declare that no competing interests exist.
  10. Marius Ueffing

    Institute for Ophthalmic Research, Center for Ophthalmology, University of Tübingen, Tübingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  11. Andrei N Lupas

    Department of Protein Evolution, Max Planck Institute for Developmental Biology, Tübingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1959-4836
  12. Christian Johannes Gloeckner

    Translational Biomarker in Neurodegenerative Diseases, German Center for Neurodegenerative Diseases, Tübingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6494-6944
  13. Marino Zerial

    Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
    For correspondence
    zerial@mpi-cbg.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7490-4235

Funding

Max-Planck-Gesellschaft (Open-access funding)

  • Marino Zerial

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

Reviewing Editor

  1. Suzanne R Pfeffer, Stanford University School of Medicine, United States

Version history

  1. Received: February 21, 2019
  2. Accepted: October 22, 2019
  3. Accepted Manuscript published: November 13, 2019 (version 1)
  4. Version of Record published: November 14, 2019 (version 2)
  5. Version of Record updated: August 18, 2022 (version 3)

Copyright

© 2019, Lauer 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

  • 1,719
    Page views
  • 244
    Downloads
  • 18
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

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. Janelle Lauer
  2. Sandra Segeletz
  3. Alice Cezanne
  4. Giambattista Guaitoli
  5. Francesco Raimondi
  6. Marc Gentzel
  7. Vikram Alva
  8. Michael Habeck
  9. Yannis Kalaidzidis
  10. Marius Ueffing
  11. Andrei N Lupas
  12. Christian Johannes Gloeckner
  13. Marino Zerial
(2019)
Auto-regulation of Rab5 GEF activity in Rabex5 by allosteric structural changes, catalytic core dynamics and ubiquitin binding
eLife 8:e46302.
https://doi.org/10.7554/eLife.46302

Share this article

https://doi.org/10.7554/eLife.46302

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Karolina Honzejkova, Dalibor Kosek ... Tomas Obsil
    Research Article

    Apoptosis signal-regulating kinase 1 (ASK1) is a crucial stress sensor, directing cells toward apoptosis, differentiation, and senescence via the p38 and JNK signaling pathways. ASK1 dysregulation has been associated with cancer and inflammatory, cardiovascular, and neurodegenerative diseases, among others. However, our limited knowledge of the underlying structural mechanism of ASK1 regulation hampers our ability to target this member of the MAP3K protein family towards developing therapeutic interventions for these disorders. Nevertheless, as a multidomain Ser/Thr protein kinase, ASK1 is regulated by a complex mechanism involving dimerization and interactions with several other proteins, including thioredoxin 1 (TRX1). Thus, the present study aims at structurally characterizing ASK1 and its complex with TRX1 using several biophysical techniques. As shown by cryo-EM analysis, in a state close to its active form, ASK1 is a compact and asymmetric dimer, which enables extensive interdomain and interchain interactions. These interactions stabilize the active conformation of the ASK1 kinase domain. In turn, TRX1 functions as a negative allosteric effector of ASK1, modifying the structure of the TRX1-binding domain and changing its interaction with the tetratricopeptide repeats domain. Consequently, TRX1 reduces access to the activation segment of the kinase domain. Overall, our findings not only clarify the role of ASK1 dimerization and inter-domain contacts but also provide key mechanistic insights into its regulation, thereby highlighting the potential of ASK1 protein-protein interactions as targets for anti-inflammatory therapy.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Daniyal Tariq, Nicole Maurici ... Brian R Crane
    Research Article

    Circadian clocks are composed of transcription-translation negative feedback loops that pace rhythms of gene expression to the diurnal cycle. In the filamentous fungus Neurospora crassa, the proteins Frequency (FRQ), the FRQ-interacting RNA helicase (FRH), and Casein-Kinase I (CK1) form the FFC complex that represses expression of genes activated by the white-collar complex (WCC). FRQ orchestrates key molecular interactions of the clock despite containing little predicted tertiary structure. Spin labeling and pulse-dipolar electron spin resonance spectroscopy provide domain-specific structural insights into the 989-residue intrinsically disordered FRQ and the FFC. FRQ contains a compact core that associates and organizes FRH and CK1 to coordinate their roles in WCC repression. FRQ phosphorylation increases conformational flexibility and alters oligomeric state, but the changes in structure and dynamics are non-uniform. Full-length FRQ undergoes liquid–liquid phase separation (LLPS) to sequester FRH and CK1 and influence CK1 enzymatic activity. Although FRQ phosphorylation favors LLPS, LLPS feeds back to reduce FRQ phosphorylation by CK1 at higher temperatures. Live imaging of Neurospora hyphae reveals FRQ foci characteristic of condensates near the nuclear periphery. Analogous clock repressor proteins in higher organisms share little position-specific sequence identity with FRQ; yet, they contain amino acid compositions that promote LLPS. Hence, condensate formation may be a conserved feature of eukaryotic clocks.