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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 / regulation of respiratory burst involved in inflammatory response / neutrophil-mediated killing of gram-positive bacterium / superoxide-generating NADPH oxidase activator activity / positive regulation of mucus secretion / hypoxia-inducible factor-1alpha signaling pathway / cellular response to L-glutamine ...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 / regulation of respiratory burst involved in inflammatory response / neutrophil-mediated killing of gram-positive bacterium / superoxide-generating NADPH oxidase activator activity / positive regulation of mucus secretion / hypoxia-inducible factor-1alpha signaling pathway / cellular response to L-glutamine / neutrophil-mediated killing of fungus / phagolysosome / positive regulation of toll-like receptor 2 signaling pathway / positive regulation of defense response to bacterium / mucus secretion / perinuclear endoplasmic reticulum / superoxide-generating NAD(P)H oxidase activity / leukotriene metabolic process / Cross-presentation of particulate exogenous antigens (phagosomes) / NADPH oxidase complex / cytochrome complex assembly / response to yeast / cellular response to testosterone stimulus / respiratory burst / WNT5:FZD7-mediated leishmania damping / respiratory burst involved in defense response / response to angiotensin / phosphatidylinositol-3,4-bisphosphate binding / ROS and RNS production in phagocytes / response to aldosterone / regulation of release of sequestered calcium ion into cytosol / hydrogen peroxide biosynthetic process / protein targeting to membrane / superoxide anion generation / small GTPase-mediated signal transduction / superoxide metabolic process / Detoxification of Reactive Oxygen Species / positive regulation of reactive oxygen species biosynthetic process / cellular response to cadmium ion / cellular response to ethanol / tertiary granule membrane / cellular response to angiotensin / RHO GTPases Activate NADPH Oxidases / monoatomic ion channel complex / RAC2 GTPase cycle / RAC3 GTPase cycle / cellular defense response / NADPH binding / specific granule membrane / stress fiber / positive regulation of superoxide anion generation / positive regulation of endothelial cell proliferation / response to nutrient / RAC1 GTPase cycle / positive regulation of smooth muscle cell proliferation / phosphatidylinositol binding / FAD binding / response to interleukin-1 / positive regulation of phagocytosis / secretory granule / epithelial cell proliferation / response to activity / small monomeric GTPase / cellular response to glucose stimulus / response to nutrient levels / establishment of localization in cell / cellular response to mechanical stimulus / cellular response to gamma radiation / defense response / SH3 domain binding / positive regulation of interleukin-6 production / VEGFA-VEGFR2 Pathway / cytoplasmic side of plasma membrane / phagocytic vesicle membrane / positive regulation of angiogenesis / positive regulation of tumor necrosis factor production / cellular response to tumor necrosis factor / nuclear envelope / flavin adenine dinucleotide binding / positive regulation of cell growth / monoatomic ion transmembrane transport / response to hypoxia / electron transfer activity / endosome / apical plasma membrane / inflammatory response / response to xenobiotic stimulus / protein heterodimerization activity / innate immune response / focal adhesion / neuronal cell body / GTPase activity / heme binding / dendrite / endoplasmic reticulum membrane / Neutrophil degranulation / GTP binding / metal ion binding / membrane / plasma membrane
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 / Rab subfamily of small GTPases / Src homology 3 domains / 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 1.1Feb 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

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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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