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- PDB-8wej: Structure of human phagocyte NADPH oxidase in the activated state -

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Basic information

Entry
Database: PDB / ID: 8wej
TitleStructure of human phagocyte NADPH oxidase in the activated state
Components
  • (Cytochrome b-245 ...) x 2
  • 7D5 Fab heavy chain
  • 7D5 Fab light chain
  • Neutrophil cytosol factor 1
  • Neutrophil cytosolic factor 2 (65kDa, chronic granulomatous disease, autosomal 2), isoform CRA_a
  • Rac family small GTPase 1
KeywordsOXIDOREDUCTASE / NOX2 / p22 / CYBA / CYBB / TP1170 / NOX / p67 / Rac / p47
Function / homology
Function and homology information


negative regulation of glomerular filtration by angiotensin / smooth muscle hypertrophy / superoxide-generating NADPH oxidase activity / Oxidoreductases; Acting on NADH or NADPH; With oxygen as acceptor / hypoxia-inducible factor-1alpha signaling pathway / regulation of respiratory burst involved in inflammatory response / superoxide-generating NADPH oxidase activator activity / cellular response to L-glutamine / phagolysosome / reactive oxygen species biosynthetic process ...negative regulation of glomerular filtration by angiotensin / smooth muscle hypertrophy / superoxide-generating NADPH oxidase activity / Oxidoreductases; Acting on NADH or NADPH; With oxygen as acceptor / hypoxia-inducible factor-1alpha signaling pathway / regulation of respiratory burst involved in inflammatory response / superoxide-generating NADPH oxidase activator activity / cellular response to L-glutamine / phagolysosome / reactive oxygen species biosynthetic process / positive regulation of toll-like receptor 2 signaling pathway / positive regulation of defense response to bacterium / perinuclear endoplasmic reticulum / NAD(P)H oxidase H2O2-forming activity / cellular response to testosterone stimulus / Cross-presentation of particulate exogenous antigens (phagosomes) / superoxide-generating NAD(P)H oxidase activity / NADPH oxidase complex / cytochrome complex assembly / cellular response to phorbol 13-acetate 12-myristate / superoxide anion generation / WNT5:FZD7-mediated leishmania damping / respiratory burst / response to angiotensin / ROS and RNS production in phagocytes / phosphatidylinositol-3,4-bisphosphate binding / hydrogen peroxide biosynthetic process / protein targeting to membrane / cellular response to ethanol / superoxide metabolic process / Detoxification of Reactive Oxygen Species / positive regulation of reactive oxygen species biosynthetic process / small GTPase-mediated signal transduction / cellular response to cadmium ion / response to aldosterone / cellular response to angiotensin / tertiary granule membrane / RAC3 GTPase cycle / RAC2 GTPase cycle / RHO GTPases Activate NADPH Oxidases / NADPH binding / cellular defense response / specific granule membrane / positive regulation of superoxide anion generation / positive regulation of endothelial cell proliferation / stress fiber / RAC1 GTPase cycle / FAD binding / positive regulation of smooth muscle cell proliferation / response to nutrient / positive regulation of phagocytosis / secretory granule / response to interleukin-1 / phosphatidylinositol binding / response to activity / cellular response to tumor necrosis factor / small monomeric GTPase / cellular response to glucose stimulus / defense response / SH3 domain binding / cellular response to mechanical stimulus / cellular response to gamma radiation / response to nutrient levels / positive regulation of interleukin-6 production / VEGFA-VEGFR2 Pathway / phagocytic vesicle membrane / positive regulation of angiogenesis / flavin adenine dinucleotide binding / positive regulation of tumor necrosis factor production / nuclear envelope / positive regulation of cell growth / G protein activity / response to hypoxia / cytoplasmic side of plasma membrane / electron transfer activity / innate immune response / endosome / inflammatory response / apical plasma membrane / response to xenobiotic stimulus / protein heterodimerization activity / focal adhesion / neuronal cell body / heme binding / Neutrophil degranulation / dendrite / GTP binding / endoplasmic reticulum membrane / membrane / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Cytochrome b245, heavy chain / Cytochrome b558 alpha-subunit / Cytochrome Cytochrome b558 alpha-subunit / Neutrophil cytosol factor 1 / Neutrophil cytosol factor 1, C-terminal / Neutrophil cytosol factor 1, PBR/AIR / Neutrophil cytosol factor 1, PX domain / Neutrophil cytosol factor 1, first SH3 domain / Neutrophil cytosol factor 1, second SH3 domain / NADPH oxidase subunit p47Phox, C terminal domain ...Cytochrome b245, heavy chain / Cytochrome b558 alpha-subunit / Cytochrome Cytochrome b558 alpha-subunit / Neutrophil cytosol factor 1 / Neutrophil cytosol factor 1, C-terminal / Neutrophil cytosol factor 1, PBR/AIR / Neutrophil cytosol factor 1, PX domain / Neutrophil cytosol factor 1, first SH3 domain / Neutrophil cytosol factor 1, second SH3 domain / NADPH oxidase subunit p47Phox, C terminal domain / Neutrophil cytosol factor 1, PBR/AIR / : / Ferric reductase, NAD binding domain / : / Ferric reductase NAD binding domain / FAD-binding 8 / FAD-binding domain / Ferric reductase transmembrane component-like domain / Ferric reductase like transmembrane component / PhoX homologous domain, present in p47phox and p40phox. / PX domain profile. / PX domain / Phox homology / PX domain superfamily / Small GTPase Rho / Small GTPase Rab domain profile. / Small GTPase Rho domain profile. / Small GTPase Ras domain profile. / FAD-binding domain, ferredoxin reductase-type / Ferredoxin-NADP reductase (FNR), nucleotide-binding domain / Ferredoxin reductase-type FAD binding domain profile. / Riboflavin synthase-like beta-barrel / SH3 domain / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family / Src homology 3 domains / Rab subfamily of small GTPases / SH3-like domain superfamily / Src homology 3 (SH3) domain profile. / SH3 domain / Small GTP-binding protein domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DIHYDROFLAVINE-ADENINE DINUCLEOTIDE / GUANOSINE-5'-TRIPHOSPHATE / PROTOPORPHYRIN IX CONTAINING FE / Chem-NDP / : / small monomeric GTPase / NADPH oxidase 2 / Cytochrome b-245 light chain / Neutrophil cytosol factor 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.79 Å
AuthorsChen, L. / Liu, X.
Funding support China, 3items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31821091 China
Ministry of Science and Technology (MoST, China)2022YFA1303000 China
National Natural Science Foundation of China (NSFC)32225027 China
CitationJournal: Nature / Year: 2024
Title: Structure of human phagocyte NADPH oxidase in the activated state.
Authors: Xiaoyu Liu / Yiting Shi / Rui Liu / Kangcheng Song / Lei Chen /
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 oxygen. This process ...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 oxygen. This process generates superoxide anions that are vital for killing pathogens. The activation of phagocyte NADPH oxidase requires membrane translocation and the binding of several cytosolic factors. 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.
History
DepositionSep 18, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 24, 2024Provider: repository / Type: Initial release
Revision 1.0Jan 24, 2024Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.1Feb 28, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Mar 20, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Oct 16, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification
Revision 1.4Aug 12, 2026Group: Data collection / Derived calculations / Structure summary
Category: em_admin / pdbx_modification_feature ...em_admin / pdbx_modification_feature / pdbx_nonpoly_atom_coordination / pdbx_nonpoly_atom_coordination_sphere / pdbx_nonpoly_atom_coordination_sphere_order
Item: _em_admin.last_update / Description: Metalloprotein remediation / Provider: repository / Type: Remediation
Revision 1.1Aug 12, 2026Data content type: EM metadata / Data content type: EM metadata / Group: Experimental summary / Data content type: EM metadata / Category: em_admin / Data content type: EM metadata / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cytochrome b-245 light chain
B: Cytochrome b-245 heavy chain
C: Neutrophil cytosol factor 1
D: Neutrophil cytosolic factor 2 (65kDa, chronic granulomatous disease, autosomal 2), isoform CRA_a
E: Rac family small GTPase 1
H: 7D5 Fab heavy chain
L: 7D5 Fab light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)233,88217
Polymers229,5157
Non-polymers4,36710
Water181
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Cytochrome b-245 ... , 2 types, 2 molecules AB

#1: Protein Cytochrome b-245 light chain


Mass: 21005.398 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CYBA / Production host: Homo sapiens (human) / References: UniProt: P13498
#2: Protein Cytochrome b-245 heavy chain


Mass: 65412.727 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CYBB / Production host: Homo sapiens (human) / References: UniProt: P04839

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Protein , 3 types, 3 molecules CDE

#3: Protein Neutrophil cytosol factor 1


Mass: 32813.297 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NCF1 / Production host: Homo sapiens (human) / References: UniProt: P14598
#4: Protein Neutrophil cytosolic factor 2 (65kDa, chronic granulomatous disease, autosomal 2), isoform CRA_a


Mass: 34978.535 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NCF2 / Production host: Homo sapiens (human) / References: UniProt: A0A024R936
#5: Protein Rac family small GTPase 1


Mass: 21463.143 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAC1 / Production host: Homo sapiens (human) / References: UniProt: A0A8C8XFZ1

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Antibody , 2 types, 2 molecules HL

#6: Antibody 7D5 Fab heavy chain


Mass: 27603.949 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Escherichia coli (E. coli)
#7: Antibody 7D5 Fab light chain


Mass: 26238.188 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Escherichia coli (E. coli)

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Sugars , 2 types, 3 molecules

#8: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#10: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 6 types, 8 molecules

#9: Chemical ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C34H32FeN4O4
#11: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#12: Chemical ChemComp-NDP / NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE


Mass: 745.421 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H30N7O17P3
#13: Chemical ChemComp-FDA / DIHYDROFLAVINE-ADENINE DINUCLEOTIDE


Mass: 787.566 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H35N9O15P2
#14: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#15: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: activated NOX2-p22 complex / Type: COMPLEX / Entity ID: #1-#7 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 52 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 2.79 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 209359 / Symmetry type: POINT

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