Abstract
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 (p40phox, p47phox, and p67phox) 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 1:1:1 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.
Highlights
Phagocyte NADPH oxidase produces superoxide anions, a precursor of reactive oxygen species (ROS) critical for host responses to microbial infections
The cytosolic subunits p40phox, p47phox, and p67phox were tagged with fluorescent proteins (FPs; Table S1), either cyan (CFP: mTurquoise or Aquamarine) [7], yellow (YFP: Citrine) [8], or red (RFP: mCherry) [9]
The ROS production started upon activation with phorbol myristate acetate (PMA) and stopped immediately after addition of diphenyleneiodonium (DPI), a NADPH oxidase inhibitor
Summary
Phagocyte NADPH oxidase produces superoxide anions, a precursor of reactive oxygen species (ROS) critical for host responses to microbial infections. The molecular crowding, the local ion concentrations, pH, or viscosity present in live cells may change the set of accessible conformations When they are associated with diseases, knowledge about both their structure and their affinity with partners is essential to identify potential drug targets in medicinal chemistry. We combined live cell FRET–FLIM5 and FCCS approaches to propose an integrated analytical workflow for structural and quantitative studies of a protein complex composed of such multidomain proteins in their native environment We applied this workflow to the intra- and intermolecular interactions of the cytosolic factors of the phagocyte NADPH oxidase complex. Using live cell FRET–FLIM and FCCS approaches with fluorescent protein-tagged subunits, we demonstrated a 1:1:1 stoichiometry of the three subunits, estimated their affinity, and analyzed their spatial organization We used these findings to elaborate a new 3D in silico model of the entire cytosolic complex in the live cell situation. It can guide the identification of potential sites for anti-inflammatory drug targets to regulate the NADPH oxidase activity
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