Human MICAL1 is a member of a recently discovered family of multidomain proteins that couple a FAD-containing monooxygenase-like domain to typical protein interaction domains. Growing evidence implicates the NADPH oxidase reaction catalyzed by the flavoprotein domain in generation of hydrogen peroxide as a second messenger in an increasing number of cell types and as a specific modulator of actin filaments stability. Several proteins of the Rab families of small GTPases are emerging as regulators of MICAL activity by binding to its C-terminal helical domain presumably shifting the equilibrium from the free – auto-inhibited – conformation to the active one. We here extend the characterization of the MICAL1–Rab8 interaction and show that indeed Rab8, in the active GTP-bound state, stabilizes the active MICAL1 conformation causing a specific four-fold increase of kcat of the NADPH oxidase reaction. Kinetic data and small-angle X-ray scattering (SAXS) measurements support the formation of a 1:1 complex between full-length MICAL1 and Rab8 with an apparent dissociation constant of approximately 8 μM. This finding supports the hypothesis that Rab8 is a physiological regulator of MICAL1 activity and shows how the protein region preceding the C-terminal Rab-binding domain may mask one of the Rab-binding sites detected with the isolated C-terminal fragment. SAXS-based modeling allowed us to propose the first model of the free full-length MICAL1, which is consistent with an auto-inhibited conformation in which the C-terminal region prevents catalysis by interfering with the conformational changes that are predicted to occur during the catalytic cycle.
Human MICAL1 : Activation by the small GTPase Rab8 and small-angle X-ray scattering studies on the oligomerization state of MICAL1 and its complex with Rab8 / A. Esposito, V. Ventura, M.V. Petoukhov, A. Rai, D.I. Svergun, M.A. Vanoni. - In: PROTEIN SCIENCE. - ISSN 0961-8368. - 28:1(2019 Jan), pp. 150-166.
|Titolo:||Human MICAL1 : Activation by the small GTPase Rab8 and small-angle X-ray scattering studies on the oligomerization state of MICAL1 and its complex with Rab8|
VANONI, MARIA ANTONIETTA (Corresponding)
|Parole Chiave:||auto-inhibition; enzyme kinetics; FAD-containing monooxygenase/oxidase; flavoprotein; MICAL; protein–protein interaction; Rab; regulation; small-angle X-ray scattering; Biochemistry; Molecular Biology|
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
|Data di pubblicazione:||gen-2019|
|Data ahead of print / Data di stampa:||31-ott-2018|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.1002/pro.3512|
|Appare nelle tipologie:||01 - Articolo su periodico|