Fig. 5: Effective relief of MICAL1 autoinhibition via dual Rab-binding to the RBD.

a–c Structural comparison of the RBD in the autoinhibited MICAL1 (a) with the isolated fragment in apo form (PDB: 5LE0) (b) or Rab-bound form (PDB: 5LPN) (c) by superimposing their H2 helices of the RBDs. Potential atomic clashes between the CH ___domain in the autoinhibited state and the H3 helix in the apo-RBD or Rab-bound RBD are indicated by dash circles, while the H3 helix in the autoinhibited RBD is missing. Two Rab10 molecules simultaneously bind to the RBD via two Rab-binding sites (RBS-I/II). The binding of Rab10 to RBS-I results in severe atomic clashes with both the CH and LIM domains in autoinhibited MICAL1. d–f Structural comparison of the H1 helix in the RBD between the autoinhibited conformation and the Rab-bound form. The rotation of H1 results in a loss of the interaction between the H1 N-terminal part and the MO ___domain. The key interface residues in the RBD and MO domains are indicated. Notably, the sidechain of F925 in the Rab-bound RBD structure is unassigned. The H2 helix in the RBD was used for structural alignments in panels (a–f). g, h NADPH-consumption measurements (g) and the statistical analysis (h) of MICAL1 and its variants (0.5 μM) with or without the addition of Rab8a (40 μM). Rab8a used hereafter is a truncation of Rab8a^1-181 with a constitutively active mutation of Q67L, except otherwise indication. The results were normalized against the activity of the MO fragment. The fold-change of enzymatic activity induced by Rab binding for each condition is calculated for comparison. Three independent repeats were performed for each condition. i Normalized intensity of pyrene-labeled F-actin disassembled by MICAL1 and its variants (20 nM) with or without the addition of Rab8a (1.6 μM). Reactions were initiated by adding MICAL protein and NADPH. Mean values from three repeats for each sample were fit with a sigmoidal model to estimate the half-time (t_1/2) for comparison. Notably, in panels (g–i), the construct labeled as 1-1055 indicates the shorter truncation MICAL1^1-1055, which deletes the C-terminal 12 residues.