Effectors of Rab GTPases: Rab Binding Specificity and Their Role in Coordination of Rab Function and Localization - Ras Superfamily Small G Proteins: Biology and Mechanisms 2 - page 49

Rab effector domains of Rabenosyn-5. Structure-based point mutations of Rab5 to
the corresponding residues in Rab4 switch I and switch II significantly reduce its
affinity to the Rabenosyn-5 HR2 domain, and Rab5-mimicking point mutations in
switch I of Rab4 reduce the affinity towards the Rabenosyn-5 HepR domain
(Eathiraj et al. 2005 ). Another example is the highly specific interaction of Rab27
with Slac2-a, an effector protein that does not interact with Rab3 that is highly
homologous to Rab27. Replacement of only four CDR1 and switch II residues in
Rab3a by the corresponding residues of Rab27b led to a significant Slac2-a binding
activity (Kukimoto-Niino et al. 2008 ), demonstrating that selectivity within highly
homologous Rab proteins can be achieved by only a couple of residues. However,
this is not the case for phylogenetically distinct Rab GTPases. Mutations of the
Rab5 switch region that exchange amino acids with their homologues in Rab4 lead
to a reduced affinity for Rab5 effectors. However, the Rab5-mimicking switch
region mutant of Rab4 is not able to interact with these effectors. Additional
substitutions in the proximal protein core are necessary to achieve binding of
these Rab4 mutants to Rab5-specific effectors (Mishra et al. 2010 ). This demon-
strates that, besides the surface residues, structure and conformational stability of
the switch regions (and therefore of the protein core) are important for specific
effector recognition (Mishra et al. 2010 ).
A comparison of different structures of Rab:effector complexes reveals that,
even though several effectors interact with Rab proteins via ʱ -helical domains, the

orientation of the -helices on the surface of the Rab is significantly different

(Fig. 3.4 ). The coiled-coil GCC185 and the Rabaptin-5 effector structures show,
for example, a rotation of approx. 90 of the axis drawn through the longitude of the
ʱ

ʱ

-helices. Even effectors binding to the same GTPase exhibit absolutely different
structures (i.e., the Rab5 binding effectors EAA1 and Rabaptin-5). However, the
Rab binding sites of most of the effectors exhibit a common feature, which can be
seen in Fig. 3.4 where we colored the interacting residues of the effector proteins
according to their properties (hydrophobic, charged, neutral). All Rab effectors
clearly display a hydrophobic patch on the surface that is involved in Rab interac-
tion. In the complexes, the hydrophobic patches interact with a complementary
hydrophobic surface on the Rab proteins, which is formed by nonpolar residues of
switch I and switch II and the conserved hydrophobic triad. This confirms the
proposed crucial role of the hydrophobic triad in specific recognition of binding
partners, as these residues can achieve specificity by structural conformation as
discussed earlier. This observation may help to identify potential Rab binding sites
for effectors of known ternary structure without determining the complex structures
as solvent-exposed hydrophobic residues have a high potential to be part of the Rab
binding interface.