Auto-regulation of Rab5 GEF activity in Rabex5 by allosteric structural changes, catalytic core dynamics and ubiquitin binding
- Max Planck Institute of Molecular Cell Biology and Genetics, Germany
- German Center for Neurodegenerative Diseases, Germany
- Heidelberg University, Germany
- Technical University Dresden, Germany
- Max Planck Institute for Developmental Biology, Germany
- Max Planck Institute for Biophysical Chemistry, Germany
- 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
Christian Johannes Gloeckner
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.
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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
Further reading
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- Structural Biology and Molecular Biophysics
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Inhibition of Bruton’s tyrosine kinase (BTK) has proven to be highly effective in the treatment of B-cell malignancies such as chronic lymphocytic leukemia (CLL), autoimmune disorders, and multiple sclerosis. Since the approval of the first BTK inhibitor (BTKi), Ibrutinib, several other inhibitors including Acalabrutinib, Zanubrutinib, Tirabrutinib, and Pirtobrutinib have been clinically approved. All are covalent active site inhibitors, with the exception of the reversible active site inhibitor Pirtobrutinib. The large number of available inhibitors for the BTK target creates challenges in choosing the most appropriate BTKi for treatment. Side-by-side comparisons in CLL have shown that different inhibitors may differ in their treatment efficacy. Moreover, the nature of the resistance mutations that arise in patients appears to depend on the specific BTKi administered. We have previously shown that Ibrutinib binding to the kinase active site causes unanticipated long-range effects on the global conformation of BTK (Joseph et al., 2020). Here, we show that binding of each of the five approved BTKi to the kinase active site brings about distinct allosteric changes that alter the conformational equilibrium of full-length BTK. Additionally, we provide an explanation for the resistance mutation bias observed in CLL patients treated with different BTKi and characterize the mechanism of action of two common resistance mutations: BTK T474I and L528W.
