The Ras superfamily of small GTPases regulate signalling pathways in living cells by oscillating between active GTP-bound forms and inactive GDP-bound states. The Rab family of proteins comprise the largest family in the Ras GTPase superfamily and play a critical role in maintaining the structure of intracellular compartments, vesicle trafficking and fusion (Bock et al.
). All Rabs are anchored to the lipid bilayer via
prenylation of C-terminal cysteine residues by geranylgeranyltransferase (GGTase) and in their active GTP-bound state Rabs localize to distinct subcellular compartments, where they carry out their specific function through the recruitment of effector proteins (Zerial & McBride, 2001
The Rab11 subfamily comprises three isoforms: Rab11a, Rab11b and Rab25. Rab11a is a 218-residue (24.4 kDa) protein involved in the regulation of endocytic recycling pathways and has also been shown to localize to the trans-Golgi network (TGN) and regulate the traffic between endosomes and the TGN (Wilcke et al.
). Recently, a novel set of effectors termed the Rab11 family of interacting proteins (Rab11-FIPs) have been identified that contain a highly conserved C-terminal Rab-binding domain (RBD) of unknown structure (Prekeris et al.
; Hales et al.
; Lindsay & McCaffrey, 2002
). Rab11 effectors are classified into different classes based upon their sequence and domain organization. All Rab11-FIPs contain an RBD close to the C-terminal end and the family is subdivided depending on the presence of either C2 domains or EF-hand motifs at their N-termini. The class 1 Rab11-FIPs include Rip11 (Rab1-interacting protein), Rab11-FIP2 and RCP (Rab11-coupling protein) and are distinguished by having C2 phospholipid-binding domains at their N-termini (Lindsay & McCaffrey, 2004
). Class 2 proteins include Rab11-FIP3/eferin and Rab11-FIP4 and these proteins contain EF-hand motifs for calcium sensing (Hales et al.
; Wallace et al.
). Rab11-FIP1 does not belong to either category and apart from the RBD lacks similarities to known proteins (Hales et al.
; Prekeris, 2003
). Previous biophysical studies of the RBD have revealed that it contains significant α-helical content and forms homodimers in solution (Junutula et al.
). Modelling studies have predicted an amphipathic α-helical coiled coil that contains a Rab-binding hydrophobic patch along one face of the helix (Junutula et al.
; Meyers & Prekeris, 2002
; Lindsay et al.
Rab11-FIP2 is a 512-residue protein that contains a C2 domain at the N-terminus (residues 1–129), a myosin Vb-binding region (129–290) and an RBD at the C-terminus (477–496). Rab11-FIP2 protein has been found to be essential for the recycling of vesicles bearing the chemokine receptor CXCR2 back to the plasma membrane and the ternary complex Rab11–Rab11-FIP2–myosin Vb is proposed to link endosomes to the cytoskeleton and regulate delivery of vesicular cargo to the plasma membrane (Hales et al.
; Lindsay & McCaffrey, 2002
; Fan et al.
). In order to understand the molecular basis for recognition by this novel Rab11 effector family, we have purified and crystallized a complex of Rab11 (residues 1–173, 19.4 kDa) with a C-terminal fragment of Rab11-FIP2 (residues 410–512, 11.9 kDa) containing the RBD. Although the conserved sequence within the Rab11-FIPs is restricted to a short 20-residue stretch, we chose a larger C-terminal segment of Rab11-FIP2 in order to encompass the entire globular region, thereby producing a soluble fragment for crystallization.