Figure 5: MICAL1 interacts with Rab35 through a flat three-helix domain.

(a) Structure of human MICAL1^918–1067 C-terminal domain involved in Rab35 binding. It consists of three amphipathic α-helices (H1, H2 and H3) connected by disordered mobile loops not visible in the structure. Two different sets of conserved and exposed residues were identified on either side of the flat α-helical structure as candidates for making direct contacts with active Rab35: Surface 1 in yellow (S1: E946, V950, E953, V971, L975 and V978) and Surface 2 in orange (S2: R1012, M1015, L1034 and V1038). The side chains of aa I1048 and R1055 essential for Rab35 interaction are displayed in red. (b) Conserved residues of MICAL and MICAL-like C-terminal domains mapped on the surface of MICAL1 H2 and H3 (see also Supplementary Fig. 4). A schematic model is also shown. (c) Electrostatic potential surface representation (contoured at ±3 kT/eV; blue/red) of the C-terminal domain of MICAL1, as calculated with APBS^69,70 and visualized with Pymol. Single mutations that abolish Rab35 binding (red labels), set of mutants involved in Rab35 binding (black labels) and E1001R mutant that is not involved in Rab35 binding (white label) are indicated. Hydrophobic residues in the central part of the potential Rab35 interaction site are labelled in grey. (d) HeLa cells transfected with GFP-MICAL1 with indicated point mutations (green) were stained with β-tubulin (red) and CHMP4B (blue). Scale bars, 10 μm. Red arrow: midbody.