Fig. 4: Disruption of both interfaces effectively releases MICAL1 autoinhibition.

a NADPH-consumption measurement of MICAL1 and its double-interface mutants. Protein concentration of 1 μM was used. Three independent repeats were performed for each condition. b Statistical analysis of the single-interface and double-interface mutants of MICAL1. The results were normalized against the activity of the MO fragment. The corresponding NADPH-consumption curves are presented in Figs. 2h, 3h, and panel (a). The dot lines in the activity bars of the L598A/F925A and Y705A/F925A mutants indicate the expected additive effects of the two corresponding single-interface mutants over the basal activity of WT. The calculated additive impact is lower than the actual activity of the double-interface mutants, suggesting that the double-interface mutants contribute a cooperative effect to the disruption of autoinhibition formation. Three independent repeats were performed for each condition. c Confocal imaging of HeLa cells expressing GFP-tagged MICAL1, MO, and the double-interface mutants. d, e Statistical analyses of cellular actin intensity (d) and cell size (e) in HeLa cells overexpressing GFP-tagged MICAL1 and its variants. Data from 30 cells were analyzed for each condition. An exception was noted for the Y705A/F925A mutant, where data from 28 cells were analyzed. Representative cell imaging data for all conditions are shown in Figs. 2i, 3i, and panel (c).