Fig. 3: MICAL1 autoinhibition relies on the binding of the CH-L2α1-LIM assembly to the CC domain.

a Single-actin filament TIRF microscopy data revealed that treatment of F-actin with purified full-length MICAL1 did not alter depolymerization rates (1.27 ± 0.61 units/s, N = 60) compared to the control (1.78 ± 0.9 units/s, N = 20). Conversely, the addition of the purified MO domain resulted in a significant increase in depolymerization rates (11.61 ± 4.91 units/s, N = 42). The addition of purified CC to the MO domain at molar ratios of MO to CC at 1:50 or 1:200 exhibited no measurable effect on the depolymerization rate (11.20 ± 4.12 units/s, N = 55 for the 1:50 ratio and 11.15 ± 4.16 units/s, N = 57 for the 1:200 ratio), suggesting that the CC domain alone does not inhibit the MO domain. MICAL1^ΔCC exhibited a lower rate of depolymerization (5.51 ± 2.73 units/s, N = 42) compared to that of the MO domain. However, the addition of purified CC to MICAL1^ΔCC significantly inhibited depolymerization rates (3.23 ± 1.71 units/s, N = 56 and 2.62 ± 1.23 units/s, N = 30 for molar ratios of 1:50 and 1:200, respectively). As a control, the addition of purified CC alone did not affect the depolymerization rate of F-actin (2.43 ± 1.55 units/s, N = 23) compared to untreated control filaments. In this experiment, the total concentrations were as follows: MICAL1, MO, and MICAL1^ΔCC at 500 nM; NADPH at 200 µM; and CC at final concentrations of 25 µM (molar ratio 1:50) and 100 µM (molar ratio 1:200). The estimated surface concentration of actin was 50 molecules/µm². Data are presented as mean values ± standard deviation of depolymerization rates of individually measured actin filaments (N). P-values were calculated using a nonparametric unpaired two-tailed t-test with Welch’s correction: MO + CC (1:50), p = 0.6678 (ns); MO + CC (1:200), p = 0.6281 (ns); MICAL1^ΔCC + CC (1:50), p < 0.0001 (****); MICAL1^ΔCC + CC (1:200), p < 0.0001 (****); Representative micrographs are shown in Fig. S6. b, c, e Data from pyrene-labeled actin depolymerization assays demonstrate overall trends that are consistent with the findings from TIRF microscopy (a). In this experiment, the concentrations were as follows: MICAL1, MO, and MICAL1^ΔCC at 200 nM; NADPH at 200 µM; CC at final concentrations of 2 µM (1:10 molar ratio), 5 µM (1:25 molar ratio), and 10 µM (1:50 molar ratio); and actin at a final concentration of 2 µM. Data are presented as mean values ± standard deviation of the three independent replicates (n = 3). d BLI binding experiments involved immobilization of biotinylated CC domain on sensor tips and analysis for binding with MO or MICAL1^ΔCC. While no measurable binding was observed for MO up to a concentration of 27 µM, MICAL1^ΔCC exhibited specific binding with a K_D of 0.58 ± 0.17 µM. The data were measured in independent duplicates. f Pyrene-labeled actin depolymerization assays showed that MICAL1^ΔMO significantly inhibited the depolymerization activity of the MO domain at all tested molar ratios, with inhibition levels similar to that observed with the full-length MICAL1. The concentrations used were as follows: MICAL1 and MO at 200 nM; NADPH at 200 µM; MICAL1^ΔMO at 1 µM (1:5 molar ratio), 2 µM (1:10 molar ratio), and 4 µM (1:20 molar ratio); and actin at 2 µM. Data are presented as mean values ± standard deviation of the three independent replicates (n = 3). g Pyrene-labeled actin depolymerization assays demonstrated that disrupting the interaction between the MO domain and the CCα1 helix releases MICAL1 autoinhibition. Specifically, substituting Arg933 in CCα1, which forms a cation-π interaction with Phe399 in the MO domain in autoinhibited MICAL1, resulted in significant F-actin depolymerization. The concentrations used were as follows: MICAL1, MO, and MICAL1^R933A at 200 nM; NADPH at 200 µM; and actin at 2 µM. Data are presented as mean values ± standard deviation of the three independent replicates (n = 3).