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Structure of human phagocyte NADPH oxidase in the activated state

Nature volume 627, pages 189–195 (2024)Cite this article

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Abstract

Phagocyte NADPH oxidase, a protein complex with a core made up of NOX2 and p22 subunits, is responsible for transferring electrons from intracellular NADPH to extracellular oxygen1. This process generates superoxide anions that are vital for killing pathogens1. The activation of phagocyte NADPH oxidase requires membrane translocation and the binding of several cytosolic factors2. However, the exact mechanism by which cytosolic factors bind to and activate NOX2 is not well understood. Here we present the structure of the human NOX2–p22 complex activated by fragments of three cytosolic factors: p47, p67 and Rac1. The structure reveals that the p67–Rac1 complex clamps onto the dehydrogenase domain of NOX2 and induces its contraction, which stabilizes the binding of NADPH and results in a reduction of the distance between the NADPH-binding domain and the flavin adenine dinucleotide (FAD)-binding domain. Furthermore, the dehydrogenase domain docks onto the bottom of the transmembrane domain of NOX2, which reduces the distance between FAD and the inner haem. These structural rearrangements might facilitate the efficient transfer of electrons between the redox centres in NOX2 and lead to the activation of phagocyte NADPH oxidase.

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Fig. 1: Overall architecture of the NOX2–p22 complex in the activated state.
Fig. 2: Interaction between p67 and the DH domain of NOX2.
Fig. 3: Interaction between Rac1 and the DH domain of NOX2.
Fig. 4: Structure of the DH domain of NOX2 in the activated state.
Fig. 5: Conformational changes in NOX2 after activation.

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Data availability

Cryo-EM maps and the atomic coordinate of the NOX2 complex in the resting state have been deposited in the Electron Microscopy Data Bank (EMDB) and PDB under the accession codes EMD-38016 and 8X2L. Cryo-EM maps and the atomic coordinate of the NOX2 complex in the activated state have been deposited in the EMDB and PDB under the accession codes EMD-37477 and 8WEJ. The entries 8GZ3, 1E96, 5O0X and 7D3F used in this study were downloaded from the PDB. Source data are provided with this paper.

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Acknowledgements

We thank previous lab member J.-X. Wu for her early work on NOX2; all of the members of the L.C. laboratory for their help; E. Pick for suggestions; J. Han for providing the cDNA for p47, p67 and Rac1; and the National Center for Protein Sciences at Peking University for assistance with negative-stain electron microscopy. Cryo-EM data collection was supported by the Electron Microscopy Laboratory and the Cryo-EM Platform at Peking University; Beijing National Laboratory for Condensed Matter Physics, the Institute of Physics, Chinese Academy of Sciences; and the Beijing branch of Songshan Lake Laboratory for Materials Science with the assistance of X. Li, Z. Guo, C. Qin, X. Pei, X. Hui, G. Wang and D. Sun. Part of the structural computation was also performed on the Computing Platform of the Center for Life Sciences and the High-Performance Computing Platform at Peking University. We thank H. Deng and M. Han in Proteinomics Facility at Technology Center for Protein Sciences, Tsinghua University, for antibody sequencing analysis of 7D5. The work was supported by grants from the Ministry of Science and Technology of China (National Key R&D Program of China, 2022YFA1303000 to L.C.), the National Natural Science Foundation of China (32225027 to L.C.) and the Center For Life Sciences (CLS to L.C.).

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Author notes

  1. These authors contributed equally: Xiaoyu Liu, Yiting Shi, Rui Liu

Authors and Affiliations

  1. State Key Laboratory of Membrane Biology, College of Future Technology, Institute of Molecular Medicine, Peking University, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Beijing, China

    Xiaoyu Liu, Yiting Shi, Rui Liu, Kangcheng Song & Lei Chen

  2. Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China

    Xiaoyu Liu & Lei Chen

  3. Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China

    Lei Chen

  4. National Biomedical Imaging Center, Peking University, Beijing, China

    Lei Chen

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Contributions

L.C. initiated the project and wrote the manuscript draft. K.S., R.L., X.L. and Y.S. screened various combinations of activators and NOX2–p22 complex. X.L. purified the protein, prepared the cryo-EM sample and collected the cryo-EM data. X.L. and L.C. processed the data and built the model. X.L., R.L. and Y.S. purified the protein sample for the activity assay and measured the activity. All authors contributed to manuscript preparation.

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Correspondence to Lei Chen.

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The authors declare no competing interests.

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Nature thanks David E. Heppner, Andrea Mattevi, Chuangye Yan and Ming Zhou for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Protein expression and purification.

a, The amino acid sequence of p22-GFP-p67. The colours of each fragment are the same as in Fig. 1a. b, The amino acid sequence of GFPnb-p47-Rac1. c, The amino acid sequence of trimera. d, Size-exclusion chromatography profile of the activated NOX2 complex in nanodisc. Fractions between dashes were used for cryo-EM sample preparation. e, Coomassie brilliant blue-stained SDS–PAGE gel of purified protein complex composed of NOX2, p22-GFP-p67, GFPnb-p47-Rac1, 7D5, and TP1170 in nanodiscs. The experiments were repeated independently twice with similar results. For gel source data, see Supplementary Fig. 1. f, Coomassie brilliant blue-stained SDS–PAGE gel of purified complex between NOX2 and p22-GFP–7D5–TP1170 protein in nanodiscs and trimera mutants. The experiments were repeated independently twice with similar results. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 2 Cryo-EM data collection and image processing.

a, Representative raw micrograph (11,146 in total) of the NOX2 complex in nanodiscs. b, 2D class averages of the NOX2 sample. c, Workflow of the cryo-EM data processing. The processing of the resting NOX2 is shown with a pink background and the processing of the activated NOX2 is shown with a light blue background.

Extended Data Fig. 3 Cryo-EM reconstructions.

a, Gold-standard Fourier shell correlation (GSFSC) curves of consensus refinement of NOX2 in resting state. b, Angular distributions of the consensus refinement of NOX2 in resting state. c, Local resolution distribution of the resting NOX2 complex after consensus refinement. d, Cut-open view of c. e, A 90°-rotated cut-open view of d. f, Local resolution distribution of the resting NOX2 complex after focused refinement. g, Cut-open view of f. h, A 90°-rotated cut-open view of g. i, Structural comparison of the resting NOX2 between the previously determined structure (green, PDB ID: 8GZ3) and the structure determined in this study (pink, PDB ID:8X2L). j, Cryo-EM density map around the FAD- and NADPH-binding site in the resting NOX2 contoured at 9.49σ. FAD is coloured in pink. The empty NADPH-binding site is circled by dash lines. k, Gold-standard Fourier shell correlation curves of consensus refinement of NOX2 in the activated state. l, Angular distributions of the consensus refinement of NOX2 in the activated state. m, Local resolution distribution of the activated NOX2 complex after consensus refinement. n, Cut-open view of m. o, A 90°-rotated cut-open view of n. p, Local resolution distribution of the activated NOX2 complex after focused refinement. q, Cut-open view of p. r, A 90°-rotated cut-open view of q.

Extended Data Fig. 4 Representative densities of NOX2 in the activated state.

a, Unsharpened consensus map of the activated NOX2 complex, contoured at 1.0σ. Regions corresponding to NOX2, p22, p67(1–219), Rac1 and p47(154–281) are coloured as in Fig. 1a. The tails of p22 and Rac1 that insert into the lipid bilayer are denoted by arrows. The zoomed-in window shows the density of the consensus map in a semi-transparent surface with the model of p47 SH3 (154–281) together with the p22 tail fitted in. b, The densities of outer haem, inner haem, FAD-Mg2+, GTP-Mg2+, NADPH, p47(149–158), p67(192–219), NOX2(2–7), NOX2(375–380), NOX2(409–412), NOX2(484–506), NOX2(536–541) and NOX2(567–570) are contoured at 11.77σ, 11.77σ, 16.42σ, 15.34σ, 4.16σ, 6.10σ, 7.56σ, 11.65σ, 5.83σ, 11.65σ, 11.65σ, 11.65σ and 8.12σ, respectively. Mg2+ ions are shown as cyan spheres.

Extended Data Fig. 5 Structural comparisons.

a, Structural comparison of the NOX2 TMD between the resting state (pink, PDB ID: 8X2L) and the activated state (light blue). Conformational changes are indicated by red arrows. The RMSD value is labelled below. b, Structural comparison between p67–Rac1 in the activated NOX2 complex (coloured) and that in the crystal structure (grey, PDB ID:1E96). p67 was used for structural alignment. c, Structural comparison between Rac1 in the activated NOX2 complex (yellow) and that in the crystal structure (grey, PDB ID: IE96). d, Structural comparison between NOX2 DH in the activated state (light blue) and csNOX5 DH (cyan, PDB ID: 5O0X). FBD was used for structural alignment. e, Structural comparison between NOX2 DH in the activated state (light blue) and DUOX1 DH in the high-calcium state (grey, PDB ID: 7D3F). FBD was used for structural alignment.

Extended Data Fig. 6 Sequence alignment.

a, Multiple sequence alignment (MSA) of DH domains of Homo sapiens NOX2 (hsNOX2, UniProt ID: P04839), Homo sapiens NOX1 (hsNOX1, UniProt ID: Q9Y5S8), Homo sapiens NOX3 (hsNOX3, UniProt ID: Q9HBY0), Danio rerio NOX2 (drNOX2, UniProt ID: Q7T2A7), Epichloe festucae NOXA (efNOXA, UniProt ID: Q2PEP0) and Epichloe festucae NOXB (efNOXB, UniProt ID: Q2PEN8). The sequences are downloaded from UniProt and aligned using ClustalX. The result of MSA is further processed in BioEdit. α-helices and β-sheets are shown as cylinders and wide arrows respectively. The conserved NADPH-interacting arginines (R356 in hsNOX2) and the C-terminal phenylalanines (F570 in hsNOX2) are boxed in blue and red, respectively. b, MSA of Homo sapiens p67 (hsP67, UniProt ID: P19878), Homo sapiens NOXA1 (hsNOXA1, UniProt ID: Q86UR1), Danio rerio p67 (drP67, UniProt ID: A2VCY2) and Epichloe festucae NOXR (efNOXR, UniProt ID: A0JC82). Conserved residues at the interfaces are highlighted in bright orange. The residues of the activation domain are presented in a red box. c, MSA of Homo sapiens Rac1 (hsRac1, UniProt ID: P63000), Homo sapiens Rac2 (hsRac2, UniProt ID: P15153), Danio rerio Rac1 (drRac1, UniProt ID: Q7ZSZ9) and Epichloe festucae RacA (efRacA, UniProt ID: A0JC80). Conserved residues at the interfaces are highlighted in bright orange.

Extended Data Fig. 7 The NADPH-binding site and F570 region.

a, The NADPH-binding site in activated NOX2. The structure of NOX2 in the activated state is shown in surface representation coloured the same as in Fig. 1. NADPH is shown as spheres. b, The activity of the NOX2 mutant R356A versus the concentration of NADPH. Data were fitted to the Michaelis–Menten equation. Each data point is shown as an open circle. The 95% confidence interval values are denoted in brackets. Data are mean ± s.d.; n = 3 technical replicates. The experiment was performed independently twice with similar results. c, The activity of the NOX2 mutants on crude cell membrane. DPI, diphenyleneiodonium, an inhibitor of NADPH oxidase. Each data point is shown as an open circle. Data are shown as mean ± standard deviations, n = 3 technical replicates. The experiment was performed independently twice with similar results. d, The activity of the NOX2 mutant F570G versus the concentration of NADPH. Data were fitted to the Michaelis–Menten equation. Each data point is shown as an open circle. The 95% confidence interval values are denoted in brackets. Data are mean ± s.d.; n = 3 technical replicates. The experiment was performed independently twice with similar results. e–h, The local cryo-EM density map of the FAD- and NADPH-binding site in four 3D classes generated from 3DVA of the activated NOX2 data (class 1, e; class 2, f; class 3, g; and class 4, h). FAD and NADPH are coloured in pink and green, respectively. The NADPH-binding site is outlined with dashes.

Source Data

Extended Data Fig. 8 Working model for NOX2 activation.

The model for phagocyte NADPH oxidase activation. NOX2, p22 and the cytosolic factors are coloured the same as in Fig. 1a. FAD, NADPH, GTP and GDP are presented as spheres. The side chain of F215 between two haems is shown as a hexagon. The side chain of F570 at the C terminus is shown as a hexagon in dashes, indicating its high structural dynamics. The electron transfer pathway is indicated with cyan. In the resting state, the DH domain of NOX2 is in the undocked conformation and NADPH binding is not stable. In the activated state, the binding of cytosolic factors promotes the docking of the DH domain onto the bottom of the TMD and also induces the contraction of the DH domain, stabilizing the binding of NADPH and bringing NADPH close to FAD. The transient displacement of the F570 side chain allows the tight packing of NADPH with FAD, enabling the efficient hydride transfer between them and the subsequent electron transfer process in NOX2.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
Full size table
Extended Data Table 2 The activation of NOX2–p22 in nanodiscs by trimera and its p67 and Rac1 mutants
Full size table

Supplementary information

Supplementary Figure 1

Uncropped SDS -PAGE gels. Cropped regions shown in Extended Data Fig. 1e, f are indicated with dashed lines.

Reporting Summary

Supplementary Video 1

Cryo-EM maps of human NOX2 in the activated state. The unsharpened consensus map coloured the same as Fig. 1a is initially shown and then the composite map is shown.

Supplementary Video 2

Structural changes of NOX2 during activation. The morph of NOX2 structure between the resting state and the activated state.

Supplementary Video 3

Structural changes of the DH domain of NOX2 during activation. The morph of the DH domain of NOX2 between the resting state and the activated state.

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Liu, X., Shi, Y., Liu, R. et al. Structure of human phagocyte NADPH oxidase in the activated state. Nature 627, 189–195 (2024). https://doi.org/10.1038/s41586-024-07056-1

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