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.
Highlights
Small GTPases were identified almost 40 years ago and the superfamily has grown to include more than 70 human members (Cherfils and Zeghouf, 2013; Rojas et al, 2012; Shih et al, 1980; Wittinghofer and Vetter, 2011)
The observed conformational differences in relative positioning between the 4 Helical Bundle (4-HB) and the Vps9 domain in Rabex5 structures (Delprato and Lambright, 2007; Delprato et al, 2004; Zhang et al, 2014), prompted us to run additional simulations allowing for rotation between the two domains forming the catalytic core
It has long been known that Rabex5 binds ubiqutin and its guanine exchange factors (GEFs) activity is stimulated by Rabaptin5 (Blumer et al, 2013; Delprato and Lambright, 2007; Horiuchi et al, 1997; Lippeet al., 2001b; Mattera and Bonifacino, 2008)
Summary
Small GTPases were identified almost 40 years ago and the superfamily has grown to include more than 70 human members (Cherfils and Zeghouf, 2013; Rojas et al, 2012; Shih et al, 1980; Wittinghofer and Vetter, 2011). These proteins regulate an array of activities such as cell growth and differentiation, organelle biogenesis, intracellular transport, cytoskeletal organization, and cell division (Cherfils and Zeghouf, 2013). Activation and deactivation of small GTPases are controlled by cycling through inactive GDP-bound and active GTP-bound states.
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