10.7554/eLife.18675.011Figure 5. The specificity of Rab proteins binding to bMERB domains.
(a) A superposition of the complex structures of Rab1:Mical-cL, Rab8:Mical-cL, Rab10:Mical-cL and Rab1R8N:Mical-cL shows that Rabs bind Micals via their N-terminus (including RabSF1), RabSF2 as well as the switch regions (Rabs are shown in cartoon representation, switch I – red, switch II – blue, RabSF1 – orange, RabSF2, magenta; Micals are shown in cartoon representation and colored in dark green (Mical-cL interacting with Rab1), yellow (Mical-cL interacting with Rab8), light green (Mical-cL interacting with Rab10) or brown (Mical-cL interacting with Rab1R8N). The sequence alignments of different Rab proteins clearly shows that the interacting residues of Rab proteins with Micals (red residues) are highly conserved (orange residues) in Rab1 and Rab8 family members (Rab1a, Rab1b, Rab35, Rab8, Rab10, Rab13, Rab15), but not in other Rabs (below the black line). (b) In all structures of Rab proteins in complex with Mical-cL, the N-termini of the Rab proteins point towards a negatively charged patch of Mical-cL (Rabs are shown in cartoon representation as above; the surface of Mical-cL is colored by charge, red – negative charge, blue – positive charge). The sequence of the N-terminal residues of each Rab protein is shown below the corresponding structure: Whereas Rab1 contains a negatively charged glutamate at position 4, Rab8 and Rab10 contain one or two lysine residues at position 3 or at position 3 and 4, respectively. Consistently, the negatively charged N-terminus of Rab1 seems to repel α-helices 1 and 2 of Mical-cL and they adopt a conformation slightly further away from Rab1 compared to Rab8 and Rab10 (also see (a)). However, after mutating the 4 N-terminal residues of Rab1 to the corresponding sequence of Rab8 (the resulting chimera is called Rab1R8N), the structure of Rab1R8N:Mical-cL shows a similar conformation of α-helices 1 and 2 as in the structure of Rab8:Mical-cL. (c) Consistently, ITC measurements show that the affinity of binding increases approximately five-fold after mutating the N-terminal residues (Rab1:Mical-cL: KD = 5.2 µM; Rab1R8N:Mical-cL: KD = 1.1 µM; Rab8:Mical-cL: KD = 0.23 µM).
DOI: http://dx.doi.org/10.7554/eLife.18675.01110.7554/eLife.18675.012Figure 5—figure supplement 1. The N-termini of Rabs determine the specificity towards bMERB domains.
(a) Schematic presentation of the interactions between Rabs and Mical-cL (Switch I and II are shown in red and blue respectively; RabSF1, RabSF2 and RabF1 are shown in orange, magenta and green; Hydrophobic interactions are indicated by black dashed lines, ionic interactions and h-bonds are indicated by orange dashed lines). (b) Mutating the four N-terminal residues of Rab1 (1-MNPE…) to the sequence corresponding to Rab8 (1-MAKT…) leads to increased binding of EHBPs: Whereas Rab1 does not form a complex with EHBP1L1 (left) and EHBP1 (second from the right), the chimeric protein Rab1R8N forms a complex with both (EHBP1L1, second from the left; EHBP1, right) as assessed by aSEC.
DOI: http://dx.doi.org/10.7554/eLife.18675.012
10.7554/eLife.18675.013Figure 5—figure supplement 2. Comparison with other Rab:effector structures.
The main interacting helix (α3) in the structure of Rab10:Mical-cL adopts a very similar relative position as the main interacting helices of the effector proteins in the structures of Rab3:Rabphilin-3a (pdb 1ZBD) (Ostermeier and Brunger, 1999), Rab27:Slp2a (pdb 3BC1) (Chavas et al., 2008) and Rab27:Slac2-a (pdb 2ZET) (Kukimoto-Niino et al., 2008). Furthermore, a basic Arg and an acidic Asp are conserved in all structures (in Rab3:Rabphilin-3a, only the Arg is conserved) and contact the residues corresponding to Asp45 and Gln61 in Rab10.
DOI: http://dx.doi.org/10.7554/eLife.18675.013