Rab GEFs and GAPs: The Enigma Variations - Ras Superfamily Small G Proteins: Biology and Mechanisms 2 - page 85

regions and amino-terminus (Kim et al. 2006 ; Cai et al. 2008 ). Bet3 and Bet5 are
both longin domain proteins and form a dimeric platform for Rab binding (Kinch
and Grishin 2006 ; Levine et al. 2013 ) (Fig. 5.2 ). Similarly to Sec2, TRAPP makes
contacts with residues in its target Rab in and adjacent to the TIGID RabF1 motif,
and the conserved tryptophan at the start of the WDTAGQE switch II sequence (Cai
et al. 2008 ). In addition, the extreme C-terminus of Bet3 has a highly conserved
di-acidic motif, DE or EE that inserts into the phosphate-binding site. The last
residue of Bet3 E192 interacts with the Rab1 P-loop lysine 21 and therefore
perturbs the interactions needed for magnesium ion and phosphate binding. In the
GEF-bound conformation the switch II region of Rab1 is restructured and the
conserved glutamine residue at position 67 rotates and also makes contact with
the P-loop lysine 21. Mutational analysis confirms that both the Bet3 acidic finger
residue E192 and Rab1 Q67 in switch II are required for TRAPP-stimulated GDP
release from Rab1 (Cai et al. 2008 ; Langemeyer et al. 2014 ). The nucleotide-free
Rab1 switch I region is only partially structured in the TRAPP complex, and the
density between residues 31 and 37 is lost (Cai et al. 2008 ). It is important to note
that switch I is not physically displaced from its normal position by TRAPP. Rather
Bet3 perturbs the interactions needed for magnesium ion and phosphate binding.
This suggests that loss of stabilisation of switch I position occurs after magnesium
ion and GDP release. Additional support for this view comes from the behaviour of
the Rab1 Q67A mutant. This shows normal GDP and GTP-binding properties, but
cannot release GDP in response to TRAPP (Langemeyer et al. 2014 ). If switch I
displacement occurred prior to switch II rearrangement, then GDP release should
have already occurred before the switch II glutamine 67 made contact with the
P-loop lysine 21.
5.2.4 DrrA: A Pathogen-Encoded Rab1 GEF
DrrA/SidM is a Legionella protein with Rab1 GDP-GTP exchange activity
required for Rab1 recruitment to the pathogen-containing intracellular vacuole
(Machner and Isberg 2006 ; Murata et al. 2006 ; Ingmundson et al. 2007 ; Machner
and Isberg 2007 ). Like the cellular GEFs discussed here, DrrA interacts with the
switch regions and P-loop of its target Rab, and therefore promotes nucleotide
exchange (Schoebel et al. 2009 ; Suh et al. 2009 ). Interestingly, there are obvious
differences in the conformation of Rab1 in Rab1-DrrA complexes when compared
to Rab1-TRAPP complexes. In the Rab1-DrrA complex the Rab1 P-loop lysine
21 contacts the Rab switch II aspartate 63 and glutamine 67. This is different to the
Rab1-TRAPP complex where no contacts between the Rab1 switch II region and
the P-loop lysine 21 are observed. It also diverges from the geometry seen in the
Ras-SOS GEF complex where the Ras P-loop lysine interacts with a conserved
glutamate intrinsic to the Ras active site switch II region (Wittinghofer and Vetter
2011 ). Mutation of this glutamate in Ras therefore reduces GEF-stimulated GDP
release (Gasper et al. 2008 ) In Rab1, an equivalent switch II glutamine mutation