Fig. 3: Suppression of MO activity and actin binding by the tripartite CH-LIM-RBD interaction.

a aSEC profiles showing no detectable binding between the fragments of MO and RBD. b, c NADPH-consumption measurements (b) and statistical analysis (c) depicting the impact of the addition of the RBD, CLR, or CLR mutant (10 μM) on the activity of the MO ___domain (0.5 μM). The V706Q/L707Q mutation was designed to destabilize the compact CLR structure, while the F925A mutation was designed to disrupt the direct interaction between the MO and CLR. The results were normalized against the activity of the MO fragment. Three independent repeats were performed for each condition. d, e aSEC profiles illustrating the interactions between the fragments of MO and CLR (d) or the V706Q/L707Q mutant (e). The backward shift of the CLR peak indicates a more compact CLR structure in the autoinhibited state. f, g The tripartite CH/LIM/RBD interface observed in the autoinhibited MICAL1 structure. h, i NADPH-consumption measurements (h) and confocal imaging of HeLa cells (i) of MICAL1 and the CH/LIM/RBD interface mutants. The protein concentration of 1 μM was used in the NADPH-consumption assay. Three independent repeats were performed for each condition in the NADPH-consumption measurements. j Quantification analysis of F-actin co-sedimentation results with the MO fragment, with or without the addition of the indicated fragments. The co-sedimentation results are presented in Supplementary Fig. 4c. Three independent repeats were performed for each condition.