NADPH Gating and F-Actin Engagement Define the Structural Bases of MICAL1 Autoinhibition and Activation

ABSTRACT: Human MICAL1 belongs to the MICAL multidomain protein family exhibiting an unprecedented [AG3.1]actin depolymerizing activity through the NADPH-dependent oxidoreductase reaction catalyzed by their N-terminal flavoprotein domain. Free MICAL1 exists in an autoinhibited state. Its relief upon interaction with activating (macro)molecules controls the timing and location of the catalytically active enzyme, explaining its participation in diverse cellular processes. To clarify the mechanisms of autoinhibition and activation of the enzyme, we combined kinetic, cryo-EM, chemical cross-linking and modeling studies on human MICAL1 forms, and their complexes with NADPH and F-actin. Our results show that MICAL1 exists in solution in a single compact conformation able to bind and oxidize NADPH, although at a slow rate. The reaction is controlled by the reorientation of the gating Trp400 residue, which is required for productive binding of the nicotinamide portion of NADPH in the active site, and is specifically hampered in the autoinhibited enzyme by the Trp400-Phe399-Arg933 triad. In the complex with F-actin, the flavoprotein domain bridges two actin subunits along the filament’s ridge. Modeling indicates that oxidation of actin residues causing filament’s destabilization most likely occurs by H2O2 diffusion from the active site, where NADPH-reduced FAD reacts with O2 , to the protein surface, rather than by direct hydroxylation of specific actin residues. Binding of full-length MICAL1 to F-actin might be made possible by a reorientation of the compact inhibitory module formed by the calponin homology, LIM and Rab binding domains relative to the catalytic domain leading to the uninhibited - also compact - conformation.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Andrea Graziadei  

LAB HEAD: Andrea Graziadei

PROVIDER: PXD077485 | Pride | 2026-10-15

REPOSITORIES: Pride