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
N -Acetylneuraminic acid (Neu5Ac) is a negatively charged nine-carbon amino sugar that is often the peripheral sugar in human cell-surface glycoconjugates. Some bacteria scavenge, import, and metabolize Neu5Ac or redeploy it on their cell surfaces for immune evasion. The import of Neu5Ac by many bacteria is mediated by tripartite ATP-independent periplasmic (TRAP) transporters. We have previously reported the structures of SiaQM, a membrane-embedded component of the Haemophilus influenzae TRAP transport system, (Currie et al., 2024). However, none of the published structures contain Neu5Ac bound to SiaQM. This information is critical for defining the transport mechanism and for further structure-activity relationship studies. Here, we report the structures of Fusobacterium nucleatum SiaQM with and without Neu5Ac. Both structures are in an inward (cytoplasmic side) facing conformation. The Neu5Ac-bound structure reveals the interactions of Neu5Ac with the transporter and its relationship with the Na+ binding sites. Two of the Na+-binding sites are similar to those described previously. We identify a third metal-binding site that is further away and buried in the elevator domain. Ser300 and Ser345 interact with the C1-carboxylate group of Neu5Ac. Proteoliposome-based transport assays showed that Ser300-Neu5Ac interaction is critical for transport, whereas Ser345 is dispensable. Neu5Ac primarily interacts with residues in the elevator domain of the protein, thereby supporting the elevator with an operator mechanism. The residues interacting with Neu5Ac are conserved, providing fundamental information required to design inhibitors against this class of proteins.
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