Structural and kinetic insights into flavin-containing monooxygenase and calponin-homology domains in human MICAL3

Abstract

MICAL is an oxidoreductase that participates in cytoskeleton reorganization via actin disassembly in the presence of NADPH. Although three MICALs (MICAL1, MICAL2 and MICAL3) have been identified in mammals, only the structure of mouse MICAL1 has been reported. Here, the first crystal structure of human MICAL3, which contains the flavin-containing monooxygenase (FMO) and calponin-homology (CH) domains, is reported. MICAL3 has an FAD/NADP-binding Rossmann-fold domain for monooxygenase activity like MICAL1. The FMO and CH domains of both MICAL3 and MICAL1 are highly similar in structure, but superimposition of the two structures shows a different relative position of the CH domain in the asymmetric unit. Based on kinetic analyses, the catalytic efficiency of MICAL3 dramatically increased on adding F-actin only when the CH domain was available. However, this did not occur when two residues, Glu213 and Arg530, were mutated in the FMO and CH domains, respectively. Overall, MICAL3 is structurally highly similar to MICAL1, which suggests that they may adopt the same catalytic mechanism, but the difference in the relative position of the CH domain produces a difference in F-actin substrate specificity.

Structural Characteristics, Binding Partners and Related Diseases of the Calponin Homology (CH) Domain

“…MICAL1/2 contains a CH2 domain and oxidizes methionine residues of actin to disassemble F-actin into G-actin (Grintsevich et al, 2016). The MICAL1/2 CH2 domain is connected to the monooxygenase domain, and Arg530 in the CH2 domain is the key residue mediating interaction with the monooxygenase domain (Kim et al, 2020). MICAL1/2 can also regulate actin dynamics and cell morphological changes via the CH2 domain through interacting with other signaling proteins (Hung et al, 2010).…”

Biosensor-Linked Immunosorbent Assay for the Quantification of Methionine Oxidation in Target Proteins

“…Next, we applied BLISA to various protein substrates with various oxidation levels; calmodulin, IDLO, LegP, and Sacde were oxidized by short-term exposure to H 2 O 2 (10,20,30, and 40 mM) and used to coat plates. The RFI for all proteins [calmodulin (Figure 4B), IDLO (Figure 4C), LegP (Figure 4D), and Sacde (Figure 4E)] gradually increased as the concentration of H 2 O 2 used to oxidize proteins increased.…”

“…Recombinant mouse MICAL1 and the oxidized form of F-actin were prepared as described previously . MICAL1-oxidized F-actin and actin were coated on black immunoplates and incubated for 2 h at room temperature and then blocked with the SuperBlock.…”

NADPH-dependent oxidation of CRMP2 through a MICAL1-Prx1 redox relay controls neurite outgrowth

“…These redox modifications of actin were implied in axonal outgrowth, neuronal plasticity, cell division, and cancer progression 30,40,41 . The oxidation of actin by MICAL-MO was enhanced when the following CH domain was included in the recombinant protein 42,43 . We confirm the higher catalytic efficiency of this construct for the oxidation of the Prxs as well (Fig.…”