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. 2022 Nov 22;11:e83743. doi: 10.7554/eLife.83743

Figure 1. The structure of the human NOX2-p22 complex in the resting state.

(A) Schematic of the NOX2 enzymatic assay. O2- produced by NOX2 are converted into H2O2 by SOD. In the presence of H2O2, horseradish peroxidase (HRP) converts the nonfluorescent Amplex Red into resorufin, the fluorescence of which is measurable and proportional to the concentration of H2O2. (B) The activity of the NOX2-p22 complex in crude cell membrane measured using Amplex Red assay. The activity is determined by subtracting the background of the enzymatic assay buffer without crude cell membrane. Data are shown as means ± standard deviations; N=3 technical replicates. (C) The amounts of O2- produced by NOX2-p22-7D5-TP1170 complex in nanodisc in the presence of Trimera are plotted versus time. The represented data are shown. DPI, diphenyliodonium, NADPH oxidase inhibitor. (D) The rates of O2- production in C are summarized. Data are shown as means ± standard deviations; N=3 technical replicates. (E) Side view of the cryo-EM map of NOX2-p22-7D5-TP1170 complex. The approximate boundaries of the phospholipid bilayer are indicated as gray thick lines. p22 and NOX2 are colored in orange and light blue. 7D5 and TP1170 are colored in gray and light green. FAD, lipid, and glycosylation decoration are colored in pink, cyan, and gold, respectively. (F) A 90°-rotated side view compared to D. (G) The cut-open view of the cryo-EM map of NOX2-p22. Haem and FAD is colored in magenta and pink, respectively. (H) A 90°-rotated bottom view of E. (I) Topology of p22 and NOX2 subunits. Transmembrane helices are shown as cylinders, unmodeled disordered regions are shown as dashed lines. The phospholipid bilayer is shown as gray layers. Glycosylation sites were indicated by protrusions. DH, dehydrogenase domain of NOX2. (J) Structure of NOX2-p22 complex in cartoon representation. The colors are the same as in D.

Figure 1.

Figure 1—figure supplement 1. Protein purification.

Figure 1—figure supplement 1.

(A) Size-exclusion chromatography profile of the NOX2-p22-7D5-TP1170 complex in nanodisc. Fractions between dashes were used for cryo-EM sample preparation. (B) Silver-stained SDS-PAGE gel of purified NOX2-p22-7D5-TP1170 protein in the nanodisc. Fractions with numbers in red were used for cryo-EM sample preparation. For uncropped gel, please see Figure 1—figure supplement 1—source data 1.
Figure 1—figure supplement 1—source data 1. The uncropped and unedited gels for Figure 1—figure supplement 1B.
Figure 1—figure supplement 2. Image processing.

Figure 1—figure supplement 2.

(A) Representative raw micrograph of NOX2-p22-7D5-TP1170 complex in nanodisc. (B) Two-dimensional (2D) class averages of NOX2-p22-7D5-TP1170 complex. (C) Workflow of the cryo-EM data processing. (D) Angular distributions of the final consensus reconstruction. (E) Gold-standard Fourier shell correlation curves of the final consensus refinement. (F) Gold-standard Fourier shell correlation curves of the focused refinement of the TP1170-constant region of Fab fragment (CH1 + CL + TP1170) using mask 1. (G) Gold-standard Fourier shell correlation curves of the focused refinement of the dehydrogenase (DH) domain using mask 2. (H) Gold-standard Fourier shell correlation curves of the focused refinement of the core domain using mask 3. (I) Local resolution of the NOX2-p22-7D5-TP1170 complex after consensus refinement. (J) Cut-open view of I. (K) Local resolution of the NOX2-p22-7D5-TP1170 complex after focused refinement. (L) Cut-open view of K.
Figure 1—figure supplement 3. Representative local electron density maps contoured at 4.62σ.

Figure 1—figure supplement 3.

(A) Local electron density maps of NOX2. (B) Local electron density maps of p22. (C) Local electron density of POPC. (D) Local electron density maps of haems bound in NOX2. (E) Local electron density map of FAD bound in FAD-binding sub-domain (FBD) of NOX2.
Figure 1—figure supplement 4. Sequence alignment of the NADPH oxidases (NOX) subunit.

Figure 1—figure supplement 4.

The sequences of the Homo sapiens NOX2 (hNOX2, UniProtKB: P04839), H. sapiens NOX1 (hNOX1, UniProtKB: Q9Y5S8), H. sapiens NOX3 (hNOX3, UniProtKB: Q9HBY0), H. sapiens NOX4 (hNOX4, UniProtKB: Q9NPH5), Cylindrospermum stagnale NOX5 (csNOX5, UniProtKB: K9WT99) and H. sapiens DUOX1 (hDUOX1, UniProtKB: Q9NRD9) were aligned. The sequence alignment of Figure 1—figure supplements 4 and 5 are all shown as follows: Conserved residues are highlighted in gray. Secondary structures are indicated as cylinders (α helices), arrows (β sheets), and lines (loops). Unmodeled residues are indicated as dashed lines. The color of arrows and cylinders is the same as in Figure 1I. Residues interact with p22 are indicated as pink-filled circles.
Figure 1—figure supplement 5. Sequence alignment of p22 subunit.

Figure 1—figure supplement 5.

The sequences of the Homo sapiens p22 (hp22, UniProtKB: P13498), Mus musculus p22 (mp22, UniProtKB: Q61462), Bos taurus p22 (bp22, UniProtKB: O46521), Danio rerio p22 (drp22, UniProtKB: Q6PH62), and Xenopus laevis p22 (xlp22, UniProtKB: Q6AZJ1) were aligned. Residues interacting with NOX2 are indicated as pink-filled circles. The blue-filled circles indicate the residues that form a polar interaction network.
Figure 1—figure supplement 6. Structural alignment of NADPH oxidases (NOX) family proteins.

Figure 1—figure supplement 6.

(A) Structural comparison of the transmembrane domain (TMD) of hNOX2 (light blue) and csNOX5 (gray). (B) Structural comparison of the dehydrogenase (DH) domain of hNOX2 (light blue) and csNOX5 (gray). (C) Structural comparison of the TMD of hNOX2 (light blue) and hDUOX1 (pink). (D) Structural comparison of the DH domain of hNOX2 (light blue) and hDUOX1 (pink).
Figure 1—figure supplement 7. Locations of chronic granulomatous disease (CGD) mutations found in human patients.

Figure 1—figure supplement 7.

(A) Mutations of NOX2 found in CGD patients (annotated in UNIPROT) are mapped onto the structural model of NOX2. Cα positions of mutations are shown as red spheres, NOX2 is shown in a light blue cartoon. (B) Mutations of p22 found in CGD patients (annotated in UNIPROT) are mapped onto the structural model of p22. Cα positions of mutations are shown as red spheres. p22 is shown in a bright orange cartoon. Mutations mentioned in the text are highlighted in bold.