Quantitative live-cell imaging and 3D modeling reveal critical functional features in the cytosolic complex of phagocyte NADPH oxidase

Cornelia S Ziegler (1) , Leila Bouchab (1) , Marc Tramier (2) , Dominique Durand (3) , Franck Fieschi (4) , Sophie Dupré-Crochet (1) , Fabienne Mérola (1) , Oliver Nüsse (1) , Marie Erard (1)

1 LCPO - Laboratoire de Chimie Physique D'Orsay
2 IGDR - Institut de Génétique et Développement de Rennes
3 I2BC - Institut de Biologie Intégrative de la Cellule
4 IBS - UMR 5075 - Institut de biologie structurale

Phagocyte NADPH oxidase produces superoxide anions, a precursor of reactive oxygen species (ROS) critical for host responses to microbial infections. However, uncontrolled ROS production contributes to inflammation, making NADPH oxidase a major drug target. It consists of two membranous (Nox2 and p22phox) and three cytosolic subunits (p40, p47, and p67) that undergo structural changes during enzyme activation. Unraveling the interactions between these subunits and the resulting conformation of the complex could shed light on NADPH oxidase regulation and help identify inhibition sites. However, the structures and the interactions of flexible proteins comprising several well-structured domains connected by intrinsically disordered protein segments are difficult to investigate by conventional techniques such as X-ray crystallography, NMR, or cryo-EM. Here, we developed an analytical strategy based on FRET-fluorescence lifetime imaging (FLIM) and fluorescence cross-correlation spectroscopy (FCCS) to structurally and quantitatively characterize NADPH oxidase in live cells. We characterized the inter- and intramolecular interactions of its cytosolic subunits by elucidating their conformation, stoichiometry, interacting fraction, and affinities in live cells. Our results revealed that the three subunits have a 111 stoichiometry and that nearly 100% of them are present in complexes in living cells. Furthermore, combining FRET data with small-angle X-ray scattering (SAXS) models and published crystal structures of isolated domains and subunits, we built a 3D model of the entire cytosolic complex. The model disclosed an elongated complex containing a flexible hinge separating two domains ideally positioned at one end of the complex and critical for oxidase activation and interactions with membrane components.

Mots clés: FRET-FLIM; FCCS; fluorescent protein; protein complex; intrinsically disordered proteins; fluorescence correlation spectroscopy (FCS); structural model; protein-protein interaction; fluorescence resonance energy transfer (FRET); unstructured domain; quaternary structure; NOX family; NADPH oxidase

Domaines: Biologie structurale [q-bio.BM]

Cornelia S Ziegler, Leila Bouchab, Marc Tramier, Dominique Durand, Franck Fieschi, et al.. Quantitative live-cell imaging and 3D modeling reveal critical functional features in the cytosolic complex of phagocyte NADPH oxidase. Journal of Biological Chemistry, 2019, 294 (11), pp.3824-3836. ⟨10.1074/jbc.RA118.006864⟩. ⟨hal-02020673⟩