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.

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

  • 1,892
    views
  • 262
    downloads
  • 35
    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. 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
    Joar Esteban Pinto Torres, Mathieu Claes ... Yann G-J Sterckx
    Research Article

    African trypanosomes are the causative agents of neglected tropical diseases affecting both humans and livestock. Disease control is highly challenging due to an increasing number of drug treatment failures. African trypanosomes are extracellular, blood-borne parasites that mainly rely on glycolysis for their energy metabolism within the mammalian host. Trypanosomal glycolytic enzymes are therefore of interest for the development of trypanocidal drugs. Here, we report the serendipitous discovery of a camelid single-domain antibody (sdAb aka Nanobody) that selectively inhibits the enzymatic activity of trypanosomatid (but not host) pyruvate kinases through an allosteric mechanism. By combining enzyme kinetics, biophysics, structural biology, and transgenic parasite survival assays, we provide a proof-of-principle that the sdAb-mediated enzyme inhibition negatively impacts parasite fitness and growth.

    1. Structural Biology and Molecular Biophysics
    Manming Xu, Sarath Chandra Dantu ... Shozeb Haider
    Research Article

    The relationship between protein dynamics and function is essential for understanding biological processes and developing effective therapeutics. Functional sites within proteins are critical for activities such as substrate binding, catalysis, and structural changes. Existing computational methods for the predictions of functional residues are trained on sequence, structural, and experimental data, but they do not explicitly model the influence of evolution on protein dynamics. This overlooked contribution is essential as it is known that evolution can fine-tune protein dynamics through compensatory mutations either to improve the proteins’ performance or diversify its function while maintaining the same structural scaffold. To model this critical contribution, we introduce DyNoPy, a computational method that combines residue coevolution analysis with molecular dynamics simulations, revealing hidden correlations between functional sites. DyNoPy constructs a graph model of residue–residue interactions, identifies communities of key residue groups, and annotates critical sites based on their roles. By leveraging the concept of coevolved dynamical couplings—residue pairs with critical dynamical interactions that have been preserved during evolution—DyNoPy offers a powerful method for predicting and analysing protein evolution and dynamics. We demonstrate the effectiveness of DyNoPy on SHV-1 and PDC-3, chromosomally encoded β-lactamases linked to antibiotic resistance, highlighting its potential to inform drug design and address pressing healthcare challenges.