Structure of the core human NADPH oxidase NOX2

Abstract

NOX2 is the prototypical member of the NADPH oxidase NOX superfamily and produces superoxide (O₂^•-), a key reactive oxygen species (ROS) that is essential in innate and adaptive immunity. Mutations that lead to deficiency in NOX2 activity correlate with increased susceptibility to bacterial and fungal infections, resulting in chronic granulomatous disease. The core of NOX2 is formed by a heterodimeric transmembrane complex composed of NOX2 (formerly gp91) and p22, but a detailed description of its structural architecture is lacking. Here, we present the structure of the human NOX2 core complex bound to a selective anti-NOX2 antibody fragment. The core complex reveals an intricate extracellular topology of NOX2, a four-transmembrane fold of the p22 subunit, and an extensive transmembrane interface which provides insights into NOX2 assembly and activation. Functional assays uncover an inhibitory activity of the 7G5 antibody mediated by internalization-dependent and internalization-independent mechanisms. Overall, our results provide insights into the NOX2 core complex architecture, disease-causing mutations, and potential avenues for selective NOX2 pharmacological modulation.

Fig. 1. Architecture of the NOX2 core-Fab 7G5 complex.

a Cryo-EM map (left) and three-dimensional reconstruction (right) of the NOX2 core-Fab 7G5 complex viewed parallel to the membrane plane, with approximate membrane boundaries indicated in gray. b Cartoon topology model of the NOX2 core complex, with the membrane bilayer colored light gray. c Model of the NOX2 core (left) and cartoon representation of the transmembrane helices (right), viewed perpendicular to the membrane from the extracellular side. d Close-up view of the NOX2 TM core, with hemes (yellow) coordinated by conserved histidine residues (blue). The metal-to-metal distance between the two hemes is 19.8 Å, with a closest interatomic distance (vinyl-to-vinyl) of 7.4 Å. The putative reduction center, composed of His115 (blue) and His119, Arg54 (orange) is located close to the p7’ propionate group of the outer heme. Hydrophobic residues located between the two heme molecules, including Phe215, are highlighted in green.

Fig. 2. The extracellular loop cap structure of NOX2.

a Close-up view of the NOX2 ECLs, with loops A, C, and E colored purple, cyan, and blue, respectively. The TMD of NOX2 is colored light blue, while p22 is shown as light red transparent surface. Glycans on loop C (Asn132 and Asn149) and loop E (Asn240) are represented as light gray sticks. The small hexagon labels indicate zoom-in views shown in panel b. b Close-up view of hydrophobic and polar interactions that contribute to the ECL fold, with loops and sidechains colored as in a. Top panel: polar intra-loop electrostatic interactions that stabilize loop C. Bottom left panel: residues that contribute to hydrophobic packing between the ECLs. Bottom right panel: electrostatic interaction between loop A residue Tyr41 and loop E residue His239, both conserved in NOX1–4. c Hydrogen bonds between the outer heme and the backbone of loop E residues. The p6’ propionate group contacts Arg226 and Val228, while the p7’ propionate group contacts Met268. Arg229, conserved in NOX1/3, contacts Tyr152 in loop C and connects the outer heme to the ECL A/C/E cap. d Putative tunnels that illustrate O₂/O₂^•- entry and exit, with putative reduction center residues colored orange. Panel 1 shows the first tunnel (green), lined by Tyr29, Tyr33 on TM1 and Asn265, Met268 on loop E. Panel 2 shows the second tunnel (red), lined by Lys38, Phe39 on loop A and Arg229, Asn265 on loop E. Sidechains that line the tunnels are represented as blue sticks.

Fig. 3. The architecture of p22 and the NOX2–p22 interface.

a Structure model of p22 (red ribbon, with the C-terminus colored orange) associating with NOX2 (blue surface), viewed parallel to the membrane. Approximate membrane boundaries are indicated in gray and interface lipids are highlighted in green. Hexagon labels refer to zoom-in views shown in panel d. The buried surface area between p22 and NOX2 is ~7400 Ų. b Structure of p22 and NOX2 viewed perpendicular to the membrane. c Close-up view of the p22 core at the intracellular side, highlighting bulkier sidechains that contribute to hydrophobic packing and form polar interactions. The C-terminus of p22 (orange) contacts the p22 core via Trp131. d Close-up view of key interaction sites at the NOX2–p22 interface. Colored hexagon labels indicate the location of each site in panel (a). Top left: on p22 ECL1 residue Thr34 forms a hydrogen bond with NOX2 TM3 residue Glu124. The ECL2 of p22 does not extend outside the membrane and is capped by NOX2, where p22 residue Leu105 contributes to a hydrophobic patch with NOX2. Right: a lipid molecule, represented in green stick with gray mesh, is wedged between p22 and NOX2. Residues at this interface (p22:Ala16, Ile20; NOX2:Trp106, Ala109) form a shape-complementing surface for the bound lipid. Residues Lys195, Arg198, and Arg199 on NOX2 ICL2 create a positively charged patch that may favor a negatively charged phospholipid headgroup. Bottom: residues in the conserved TXXT-motif (Thr191, Ser192, Ser192, Thr194 in NOX2) form hydrogen bonds with sidechains on the N-terminus of p22 (Trp9, Gln13). e Two lipid molecules (green stick with gray mesh) are observed between p22 ECL1 and NOX2 loop A.

Fig. 4. Structural and functional characterization of the anti-NOX2 antibody 7G5.

a Cryo-EM map of the NOX2 core-Fab 7G5 complex. The Fab 7G5 (colored light orange and yellow) binds within a collar formed by three distinct glycosylation sites (colored magenta). b Close-up view of the NOX2-Fab 7G5 binding interface (Fab heavy chain in yellow and light chain in light orange), highlighting the small disulfide-capped epitope in loop E (blue). c Key interactions between NOX2 and Fab 7G5. Three lysine residues and a glutamate in NOX2 loop E form hydrogen bonds and salt bridges with negatively charged and polar residues in Fab 7G5. The left and right panels highlight key interactions between NOX2 and the heavy and light chain of 7G5, respectively. d Extracellular ROS production assay on COS7 cells expressing the recombinant NOX2 enzymatic complex. Dose-dependent inhibition is observed upon the addition of 7G5 IgG (green), but not with 7G5 Fab (orange). Data represent two replicates per condition with line representing the mean value. Each data point was generated by taking the area under the curve (AUC) from a ROS production assay illustrated in Supplementary Fig. 8c. The amount of ROS in the ROS production assay was measured by relative light unit (RLU). The blue dashed line represents ROS inhibition by apocynin as illustrated in Supplementary Fig. 8b. ROS production in untreated cells without and with PMA stimulation is shown by downward (four replicates) and upward (four replicates) triangles, respectively. e, f Extracellular ROS production assay on HL60 cells (e) and human neutrophils (f) expressing endogenous NOX2. The 7G5 IgG shows inhibition of ROS production while the 7G5 Fab shows weak or no inhibition. Data were generated the same way as explained in d. g The 7G5 IgG, but not the Fab, induces NOX2 internalization. Data represent the normalized mean fluorescence intensity (MFI) with three replicates per condition, and error bars represent standard deviation (SD). MFIs were normalized to the samples with sodium azide for either the IgG or Fab, respectively. Source data are available in the Source Data File.

Fig. 5. CGD mutations in the NOX2 core complex.

Reported missense mutations in p22, NOX2 TMD and NOX2 ECL that lead to chronic granulomatous disease (CGD) are highlighted in green, orange, and pink, respectively. The center panel shows an overall view, with CGD mutations shown as spheres. Side panels provide a close-up view of selected mutations. Sidechains not reported as CGD mutations, but that are part of relevant interactions or the nearby chemical environment are colored light red for p22 and blue for NOX2. a Missense mutations Leu105Arg and Leu120Pro are predicted to disrupt the hydrophobic patch at the p22 ECL-NOX2 interface, and mutation Trp125Cys is expected to disrupt interaction with two lipids found at the interface. b The conserved loop E disulfide bond would be disrupted by mutations Cys244Ser/Arg/Gly/Tyr and Cys257Arg/Ser. Missense mutations Leu45Arg, Leu141Pro, Leu144Pro, and Leu153Arg are predicted to disrupt the hydrophobic packing in the ECLs, and the Tyr41Asp mutation would break an important hydrogen bond that connects loop A and E. c The Glu53Val/Gln, Arg90Trp/Gly/Gln/Pro, His94Arg, and Tyr121His missense mutations are predicted to destabilize the core of p22 and subsequently disrupt NOX2–p22 association. d Missense mutations Thr191Ser and Ser193Pro/Phe in the conserved ¹⁹¹TXXT¹⁹³-motif and Arg198Trp, conserved in NOX1–5 and DUOX1/2, would disrupt important interactions between NOX2 and p22, potentially destabilizing the structural scaffold near the inner heme.