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

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.

Small GTPases of the Rab and Arf Families: Key Regulators of Intracellular Trafficking in Neurodegeneration

“…Apart from its catalytic domain, Rabex5 contains a Rabaptin5 binding site, which is a Rab5 effector molecule. Thus, Rabex5 binds tightly to Rab5-regulated Rabaptin5, which in turn regulates Rabex5 GEF activity, forming a feedback loop [ 78 ].…”

Faculty Opinions recommendation of A non-linear system patterns Rab5 GTPase on the membrane.

“…Rabex5/Rabaptin5 is one of the best characterized GEF/effector complexes in eukaryotes. Rabex5 is a 57 kDa Vps9 domain containing GEF for Rab5 (Horiuchi et al, 1997;Delprato and Lambright, 2007;Lauer et al, 2019). Rabaptin5 is a 99 kDa protein with multiple protein-protein interaction sites that colocalizes with Rab5 on EE and is essential for endosome fusion (Stenmark et al, 1995;Horiuchi et al, 1997).…”

“…Rabaptin5 is a 99 kDa protein with multiple protein-protein interaction sites that colocalizes with Rab5 on EE and is essential for endosome fusion (Stenmark et al, 1995;Horiuchi et al, 1997). As Rabaptin5 forms a dimer in solution, the complex is a tetramer of two Rabaptin5 and two Rabex5 subunits (Lauer et al, 2019). The interaction with Rabaptin5 has been shown to increase Rabex5 GEF activity and produce structural rearrangements in Rabex5 (Delprato et al, 2004;Delprato and Lambright, 2007;Lippé et al, 2001;Horiuchi et al, 1997;Zhang et al, 2014;Lauer et al, 2019).…”

“…As Rabaptin5 forms a dimer in solution, the complex is a tetramer of two Rabaptin5 and two Rabex5 subunits (Lauer et al, 2019). The interaction with Rabaptin5 has been shown to increase Rabex5 GEF activity and produce structural rearrangements in Rabex5 (Delprato et al, 2004;Delprato and Lambright, 2007;Lippé et al, 2001;Horiuchi et al, 1997;Zhang et al, 2014;Lauer et al, 2019). By binding active Rab5, Rabaptin5 localizes the enhanced GEF activity of Rabex5 in the vicinity of active Rab5, thereby creating the positive feedback loop.…”

“…In addition, Rabex5 can be recruited to EE via binding to Ubiquitin via two distinct Ubiquitin binding domains near the N-terminus (Penengo et al, 2006). Interestingly, Ubiquitin binding enhances GEF activity toward Rab5 helping to initiate the positive feedback loop on endosomes carrying ubiquitinated cargo (Lauer et al, 2019). Blümer et al, 2013 observed that artificially targeting Rabex5 to mitochondria resulted in Rab5 recruitment to these organelles, suggesting that Rabex5 can be sufficient for localizing Rab5 to a membrane compartment.…”