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Elife. 2019 Nov 13;8. pii: e46302. doi: 10.7554/eLife.46302.

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

Author information

1
Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
2
German Center for Neurodegenerative Diseases, Tübingen, Germany.
3
Bioquant, Heidelberg University, Heidelberg, Germany.
4
Heidelberg University Biochemistry Centre (BZH), Heidelberg, Germany.
5
Molecular Analysis-Mass Spectrometry Center for Molecular and Cellular Bioengineering, Technical University Dresden, Dresden, Germany.
6
Max Planck Institute for Developmental Biology, Tuebingen, Germany.
7
Statistical Inverse Problems in Biophysics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
8
Felix Bernstein Institute for Mathematical Statistics in the Biosciences, University of Göttingen, Göttingen, Germany.
9
Center for Ophthalmology, Institute for Ophthalmic Research, University of Tübingen, 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.

KEYWORDS:

Rab5; guanine nucleotide exchange factor (GEF); hydrogen deuterium exchange mass spectrometry (HDX-MS); molecular biophysics; structural biology

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