Rab GEFs and GAPs: The Enigma Variations - Ras Superfamily Small G Proteins: Biology and Mechanisms 2 - page 91

5.3.1 Rab GAP Mechanism

Pioneering structural studies of Rab GAPs have revealed that the catalytic mech-
anism for GTP hydrolysis differs from that used by Ras and Rho GAPs (Pan
et al. 2006 ; Gavriljuk et al. 2012 ). For Ras, GTP hydrolysis requires a glutamine
residue intrinsic to the Ras switch II DxxGQ sequence and a catalytic arginine
finger residue from the GAP inserted into the Ras nucleotide-binding site. By
contrast the TBC1 domain contains two signature motifs IxxDxxR and YxQ
(Neuwald 1997 ), and both these are required for catalytic activity (Pan
et al. 2006 ). Structures of Rab33 GDP-AlF 3 with the Gyp1 TBC1 domain and
Rab1 GDP-BeF 3 with TBC1D20 reveal a dual finger mechanism involving the
conserved arginine of the IxxDxxR motif, and the conserved glutamine of the YxQ
motif (Pan et al. 2006 ; Gavriljuk et al. 2012 ) (Fig. 5.3 ). While the TBC1 domain
arginine adopts an equivalent position to that seen for Ras GAPs, the Rab switch II
glutamine plays no direct role in (GAP stimulated) GTP hydrolysis. Instead this
function is taken by the conserved glutamine of the YxQ motif in the TBC1 domain.
The Rab switch II glutamine projects away from the nucleotide-binding site and
contacts the backbone of the GAP. Fitting with the idea that the switch II glutamine
plays little direct role in GTP hydrolysis, mutation in this residue had little effect on
the GAP activity of RUTBC3 towards Rab5 or RUTBC1 towards Rab33B (Not-
tingham et al. 2011 ). In the case of Rab5 the switch II glutamine resulted in a
greater than 5-fold reduction in basal GTP hydrolysis (Langemeyer et al. 2014 ).
This indicates that for some Rabs basal and GAP-stimulated GTP hydrolysis may
occur via different mechanisms. Analysis of two other Rab-GAP pairs complicates
this simple picture. Rab35 and Rab1 switch II glutamine mutants showed only a
slight decrease in basal GTP hydrolysis, while GTP hydrolysis was greatly reduced
when stimulated by TBC1D10A or TBC1D20, respectively (Langemeyer
et al. 2014 ). By contrast, mutations of the catalytic arginine or glutamine residues
in the TBC domain greatly reduced the catalytic efficiency of the respective GAPs
(Pan et al. 2006 ; Haas et al. 2005 ; Haas et al. 2007 ). Thus, the major determinants of
GAP-stimulated GTP hydrolysis are the catalytic residues contributed by the GAP,
while the Rab switch II glutamine plays a greater or lesser role depending on the
Rab under scrutiny.
As already described, unlike Ras the Rabs don't always require the conserved
glutamine for hydrolysis, but may need it for the GEF reaction in some instances.
This suggests there is a fundamental difference in the conformational plasticity in
the switch regions of Ras and Rabs that has consequences for

regulatory

mechanisms.