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- PDB-7u8g: Cryo-EM structure of the core human NADPH oxidase NOX2 -

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Basic information

Entry
Database: PDB / ID: 7u8g
TitleCryo-EM structure of the core human NADPH oxidase NOX2
Components
  • 7G5 - heavy chain
  • 7G5 - light chain
  • Cytochrome b-245 light chain
  • EGFP, Cytochrome b-245 heavy chain chimera
KeywordsOXIDOREDUCTASE / heterodimer / complex / NADPH oxidase / enzyme
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 / positive regulation of mucus secretion / hypoxia-inducible factor-1alpha signaling pathway / cellular response to L-glutamine / positive regulation of toll-like receptor 2 signaling pathway / positive regulation of defense response to bacterium / mucus secretion ...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 / positive regulation of mucus secretion / hypoxia-inducible factor-1alpha signaling pathway / cellular response to L-glutamine / positive regulation of toll-like receptor 2 signaling pathway / positive regulation of defense response to bacterium / mucus secretion / perinuclear endoplasmic reticulum / Cross-presentation of particulate exogenous antigens (phagosomes) / cytochrome complex assembly / NADPH oxidase complex / superoxide-generating NAD(P)H oxidase activity / respiratory burst / WNT5:FZD7-mediated leishmania damping / response to angiotensin / response to aldosterone / regulation of release of sequestered calcium ion into cytosol / ROS and RNS production in phagocytes / hydrogen peroxide biosynthetic process / superoxide anion generation / superoxide metabolic process / Detoxification of Reactive Oxygen Species / positive regulation of reactive oxygen species biosynthetic process / cellular response to cadmium ion / tertiary granule membrane / cellular response to angiotensin / cellular response to ethanol / monoatomic ion channel complex / RAC2 GTPase cycle / RHO GTPases Activate NADPH Oxidases / RAC3 GTPase cycle / NADPH binding / specific granule membrane / positive regulation of superoxide anion generation / stress fiber / positive regulation of endothelial cell proliferation / RAC1 GTPase cycle / positive regulation of smooth muscle cell proliferation / FAD binding / response to nutrient / response to interleukin-1 / positive regulation of phagocytosis / secretory granule / bioluminescence / response to activity / generation of precursor metabolites and energy / response to nutrient levels / cellular response to glucose stimulus / establishment of localization in cell / cellular response to mechanical stimulus / defense response / cellular response to gamma radiation / SH3 domain binding / positive regulation of interleukin-6 production / VEGFA-VEGFR2 Pathway / 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 / response to xenobiotic stimulus / inflammatory response / protein heterodimerization activity / innate immune response / focal adhesion / neuronal cell body / heme binding / dendrite / Neutrophil degranulation / endoplasmic reticulum membrane / metal ion binding / membrane / plasma membrane
Similarity search - Function
Cytochrome b245, heavy chain / Cytochrome b558 alpha-subunit / Cytochrome Cytochrome b558 alpha-subunit / 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 ...Cytochrome b245, heavy chain / Cytochrome b558 alpha-subunit / Cytochrome Cytochrome b558 alpha-subunit / 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 / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / 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 / Immunoglobulins / Immunoglobulin-like / Sandwich / Mainly Beta
Similarity search - Domain/homology
PROTOPORPHYRIN IX CONTAINING FE / Chem-POV / EGFP / NADPH oxidase 2 / Cytochrome b-245 light chain
Similarity search - Component
Biological speciesHomo sapiens (human)
Oryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsNoreng, S. / Ota, N. / Sun, Y. / Masureel, M. / Payandeh, J. / Yi, T. / Koerber, J.T.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2022
Title: Structure of the core human NADPH oxidase NOX2.
Authors: Sigrid Noreng / Naruhisa Ota / Yonglian Sun / Hoangdung Ho / Matthew Johnson / Christopher P Arthur / Kellen Schneider / Isabelle Lehoux / Christopher W Davies / Kyle Mortara / Kit Wong / ...Authors: Sigrid Noreng / Naruhisa Ota / Yonglian Sun / Hoangdung Ho / Matthew Johnson / Christopher P Arthur / Kellen Schneider / Isabelle Lehoux / Christopher W Davies / Kyle Mortara / Kit Wong / Dhaya Seshasayee / Matthieu Masureel / Jian Payandeh / Tangsheng Yi / James T Koerber /
Abstract: NOX2 is the prototypical member of the NADPH oxidase NOX superfamily and produces superoxide (O), a key reactive oxygen species (ROS) that is essential in innate and adaptive immunity. Mutations that ...NOX2 is the prototypical member of the NADPH oxidase NOX superfamily and produces superoxide (O), 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.
History
DepositionMar 8, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 26, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 16, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / 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

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Assembly

Deposited unit
A: EGFP, Cytochrome b-245 heavy chain chimera
B: Cytochrome b-245 light chain
D: 7G5 - heavy chain
E: 7G5 - light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)170,00112
Polymers165,8244
Non-polymers4,1778
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein EGFP, Cytochrome b-245 heavy chain chimera / CGD91-phox / Cytochrome b(558) subunit beta / Cytochrome b558 subunit beta / Heme-binding membrane ...CGD91-phox / Cytochrome b(558) subunit beta / Cytochrome b558 subunit beta / Heme-binding membrane glycoprotein gp91phox / NADPH oxidase 2 / Neutrophil cytochrome b 91 kDa polypeptide / Superoxide-generating NADPH oxidase heavy chain subunit / gp91-1 / gp91-phox / p22 phagocyte B-cytochrome


Mass: 97669.766 Da / Num. of mol.: 1
Fragment: N-terminal 2x streptag followed by EGFP and TEV + thrombin cleavage sites.
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CYBA / Production host: Homo sapiens (human)
References: UniProt: A0A6M5E0N3, UniProt: P04839, Oxidoreductases
#2: Protein Cytochrome b-245 light chain / Cytochrome b(558) alpha chain / Cytochrome b558 subunit alpha / Neutrophil cytochrome b 22 kDa ...Cytochrome b(558) alpha chain / Cytochrome b558 subunit alpha / Neutrophil cytochrome b 22 kDa polypeptide / Superoxide-generating NADPH oxidase light chain subunit / p22 phagocyte B-cytochrome / p22-phox / p22phox


Mass: 21033.451 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

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Antibody , 2 types, 2 molecules DE

#3: Antibody 7G5 - heavy chain


Mass: 23599.393 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Oryctolagus cuniculus (rabbit) / Production host: Escherichia phage EcSzw-2 (virus)
#4: Antibody 7G5 - light chain


Mass: 23521.154 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Oryctolagus cuniculus (rabbit) / Production host: Escherichia phage EcSzw-2 (virus)

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Sugars , 1 types, 3 molecules

#6: 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.: 3 / 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 , 2 types, 5 molecules

#5: Chemical ChemComp-POV / (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / POPC


Mass: 760.076 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C42H82NO8P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
#7: Chemical ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C34H32FeN4O4 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
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: NOX2 core - Fab 7G5 complex / Type: COMPLEX / Entity ID: #1-#4 / Source: MULTIPLE SOURCES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293
Buffer solutionpH: 8 / Details: 0.03% DDM, 150 mM NaCl, 20 mM Tris
SpecimenConc.: 1.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Solarus plasma cleaner (Gatan) / Grid material: GOLD / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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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: SPOT SCAN
Electron lensMode: OTHER / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 40.4 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM softwareName: cryoSPARC / Version: 3.1.0 / Category: image acquisition
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 70486 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 31.98 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00335183
ELECTRON MICROSCOPYf_angle_d0.56297062
ELECTRON MICROSCOPYf_chiral_restr0.0509779
ELECTRON MICROSCOPYf_plane_restr0.0037852
ELECTRON MICROSCOPYf_dihedral_angle_d10.1037752

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