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

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 p22^phox) and three cytosolic subunits (p40^phox, p47^phox, and p67^phox) 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.

Keywords: FCCS; FRET–FLIM; NADPH oxidase; fluorescence correlation spectroscopy (FCS); fluorescence resonance energy transfer (FRET); fluorescent protein; intrinsically disordered proteins; protein complex; protein-protein interaction; structural model.

Figure 1.

Structural organization of the NADPH oxidase. The domain structure of the three cytosolic subunits of the phagocyte NADPH oxidase in the resting state is shown. The domains and their positions in the proteins are drawn approximately to size. Arrows denote known intramolecular interactions (blue) and intermolecular interactions (green). The dotted black line represents the possible interaction between p40^phox and p47^phox.

Figure 2.

FP-tagged subunits form a functional oxidase. A, images of triple-transfected COS^Nox2/p22 cells expressing p47^phox-CFP, p67^phox-YFP, and RFP-p40^phox subunits (left to right). Scale bar, 20 μm. Five conditions of p40^phox, p47^phox, and p67^phox with no tag, two, or three tags on either N or C terminus were tested. B, time course of the luminescence signal (L-012 + horseradish peroxidase) from triple-transfected COS^Nox2/p22 cells stimulated by PMA and stopped by the oxidase inhibitor DPI at the indicated times (condition 4, green). In orange, the signal obtained with nontransfected cells as reference. C, integrated PMA-stimulated luminescence signal over 30 min (n = 3; means ± S.E.). **, p < 0.01, Tukey's multiple comparison test.

Figure 3.

Efficient FRET between N- and C-terminal tags on all subunits (tandem). A, representative example of fluorescence decays of the tandems and their fits (dashed line). The upper panels show the residual difference plot. For p40^phox, a YFP/RFP FRET pair was used, whereas p47^phox, p67^phox, and the simple D/A tandem were labeled with a CFP/YFP FRET pair. The control shown on the left is p40^phox-YFP (τ_donor = 3.18 ns, χ² = 1.12), whereas the tandem shown on the right is RFP-p40^phox-YFP (τ_long = 3.17 ns, τ_short = 1.56 ns, accounting for 77 and 23% of the decay amplitude, respectively, χ² = 1.09). B, apparent FRET efficiencies for p40^phox, p47^phox, and p67^phox tandems and the positive control (simple D/A tandem). C, effect of p67^phox co-expression on the p47^phox tandem and of p40^phox on the p67^phox tandem. A co-transfection of the tandem with RFP alone was used as control (n = 10–30 cells; means ± S.E., raw data Fig. S2). ***, p < 0.001, Tukey's multiple comparison test.

Figure 4.

FRET reveals heterodimer formation between all subunits. A, representative fluorescence lifetime images of COS7 cells expressing p47-CFP (left panel) or p47ΔC-CFP with p67-YFP (right panel). Scale bar, 10 μm. B, corresponding histograms of fluorescence lifetimes over the image pixels. The lifetime of p47-CFP (donor alone) is 3.95 ± 0.05 ns. C–E, FRET efficiencies plotted against acceptor intensity. Each symbol represents the value for one cell. F–H, FRET efficiencies plotted against acceptor/donor ratio. Each symbol represents the mean ± S.E. of three to six cells. The dashed lines indicate the upper limit of the negative controls shown as green squares in C–E. Left panels of C and F, interaction of p47^phox and p67^phox; center panels of D and G, interaction of p40^phox and p67^phox; right panels of E and H, interaction of p40^phox and p47^phox.

Figure 5.

FCCS demonstrates that all p67^phox is in complex with p47^phox. A, example of auto-correlation and cross-correlation functions and their fits from individual cells. Shown are FCCS data from COS7 cells transfected with the p67^phox tandem (left panel) or p47^phox together with p67^phox (middle and right panels). We use the p67^phox tandem as a positive control for co-diffusion. B, fraction of protein in interaction, as a function of YFP:CFP ratio, as obtained from the correlation functions. At 2-fold excess of p47-YFP, all p67-CFP is in complex, and 50% of p47-YFP is in complex. Each symbol represents the means ± S.E. of five to ten cells from three independent experiments. We chose cells expressing YFP-tagged p47^phox in varying quantities while keeping the CFP-tagged p67^phox concentration at a nearly constant low level. This simulates the situation in neutrophils, where the expression of p47^phox is higher than p67^phox (32).

Figure 6.

Modeling of p47^phoxΔCter from SAXS data. A, experimental SAXS curve measured on p47^phoxΔCter (black dots), compared with the calculated curve (red line) obtained from the selected model shown in C using the program CRYSOL (χ² = 0.97). B, experimental SAXS curve measured on full-length p47^phox (black dots) compared with the theoretical curve (blue line) calculated from the model of p47^phox shown in Fig. 7 (χ² = 1.25). C, selected SAXS model of p47^phoxΔCter that fulfills all the criteria discussed in the text and in Fig. S6. The PX domain is in violet, the SH3 domains are in green, and the AIR domain is in cyan. Residues of the PX domain responsible for interaction with the membrane in the activated form are shown in pink.

Figure 7.

Proposed model of the complete cytosolic complex of phagocyte NADPH oxidase in the inactive state. A–C, ribbon representations of p40^phox (orange), p47^phox (red), and p67^phox (gray). The N termini are labeled in blue, and the C termini are labeled in green. Pairwise interactions are shown between p40^phox and p67^phox (A) and between p47^phox and p67^phox (B), whereas the whole 3D model of the heterotrimer is shown in C. Distances between termini, as predicted from the proposed model, are only given for model evaluation (see text). The arrow symbolizes the angular flexibility of the globular N-terminal domain of p47^phox. The stars indicate the flexible regions in p67^phox (black stars) and in p47^phox C terminus (red star). In p67^phox, blue and cyan spheres represent the Rac-binding β-hairpin insertion and the activation domains, respectively. The residues of the PX domains interacting with lipids in p40^phox are the yellow spheres. D, schematic representation of the complex showing the different functional domains.