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- PDB-8kei: Cryo-EM structure of NADPH oxidase 2 in complex with p22phox and EROS -

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Basic information

Entry
Database: PDB / ID: 8kei
TitleCryo-EM structure of NADPH oxidase 2 in complex with p22phox and EROS
Components
  • (Cytochrome b-245 ...) x 3
  • (monoclonal antibody 7G5 ...) x 2
KeywordsIMMUNE SYSTEM / NADPH oxidase
Function / homology
Function and homology information


smooth muscle hypertrophy / superoxide-generating NADPH oxidase activity / Oxidoreductases; Acting on NADH or NADPH; With oxygen as acceptor / cellular response to L-glutamine / hypoxia-inducible factor-1alpha signaling pathway / positive regulation of toll-like receptor 2 signaling pathway / respiratory burst after phagocytosis / positive regulation of defense response to bacterium / mucus secretion / perinuclear endoplasmic reticulum ...smooth muscle hypertrophy / superoxide-generating NADPH oxidase activity / Oxidoreductases; Acting on NADH or NADPH; With oxygen as acceptor / cellular response to L-glutamine / hypoxia-inducible factor-1alpha signaling pathway / positive regulation of toll-like receptor 2 signaling pathway / respiratory burst after phagocytosis / positive regulation of defense response to bacterium / mucus secretion / perinuclear endoplasmic reticulum / superoxide-generating NAD(P)H oxidase activity / Cross-presentation of particulate exogenous antigens (phagosomes) / NADPH oxidase complex / cytochrome complex assembly / respiratory burst / WNT5:FZD7-mediated leishmania damping / ROS and RNS production in phagocytes / regulation of release of sequestered calcium ion into cytosol / cellular response to ethanol / response to angiotensin / hydrogen peroxide biosynthetic process / superoxide anion generation / positive regulation of mucus secretion / superoxide metabolic process / positive regulation of reactive oxygen species biosynthetic process / cellular response to cadmium ion / response to aldosterone / Detoxification of Reactive Oxygen Species / tertiary granule membrane / RHO GTPases Activate NADPH Oxidases / monoatomic ion channel complex / RAC2 GTPase cycle / RAC3 GTPase cycle / NADPH binding / specific granule membrane / positive regulation of phagocytosis / RAC1 GTPase cycle / response to nutrient / FAD binding / secretory granule / establishment of localization in cell / defense response / 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 / nuclear envelope / flavin adenine dinucleotide binding / monoatomic ion transmembrane transport / electron transfer activity / oxidoreductase activity / endosome / inflammatory response / protein heterodimerization activity / response to xenobiotic stimulus / innate immune response / neuronal cell body / heme binding / dendrite / Neutrophil degranulation / endoplasmic reticulum membrane / endoplasmic reticulum / metal ion binding / membrane / plasma membrane
Similarity search - Function
Cytochrome b-245 chaperone 1 / Cytochrome b-245 chaperone 1 / Eros / 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 ...Cytochrome b-245 chaperone 1 / Cytochrome b-245 chaperone 1 / Eros / 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 / 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
Similarity search - Domain/homology
PROTOPORPHYRIN IX CONTAINING FE / 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine / Chem-POV / NADPH oxidase 2 / Cytochrome b-245 light chain / Cytochrome b-245 chaperone 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Oryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.56 Å
AuthorsLiang, S.Y. / Liu, A.J. / Liu, Y.Z. / Ye, R.D.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2.
Authors: Shiyu Liang / Aijun Liu / Yezhou Liu / Fuxing Wang / Youli Zhou / Yuanzhengyang Long / Tao Wang / Zheng Liu / Ruobing Ren / Richard D Ye /
Abstract: Essential for reactive oxygen species (EROS) protein is a recently identified molecular chaperone of NOX2 (gp91), the catalytic subunit of phagocyte NADPH oxidase. Deficiency in EROS is a recently ...Essential for reactive oxygen species (EROS) protein is a recently identified molecular chaperone of NOX2 (gp91), the catalytic subunit of phagocyte NADPH oxidase. Deficiency in EROS is a recently identified cause for chronic granulomatous disease, a genetic disorder with recurrent bacterial and fungal infections. Here, we report a cryo-EM structure of the EROS-NOX2-p22 heterotrimeric complex at an overall resolution of 3.56Å. EROS and p22 are situated on the opposite sides of NOX2, and there is no direct contact between them. EROS associates with NOX2 through two antiparallel transmembrane (TM) α-helices and multiple β-strands that form hydrogen bonds with the cytoplasmic domain of NOX2. EROS binding induces a 79° upward bend of TM2 and a 48° backward rotation of the lower part of TM6 in NOX2, resulting in an increase in the distance between the two hemes and a shift of the binding site for flavin adenine dinucleotide (FAD). These conformational changes are expected to compromise superoxide production by NOX2, suggesting that the EROS-bound NOX2 is in a protected state against activation. Phorbol myristate acetate, an activator of NOX2 in vitro, is able to induce dissociation of NOX2 from EROS with concurrent increase in FAD binding and superoxide production in a transfected COS-7 model. In differentiated neutrophil-like HL-60, the majority of NOX2 on the cell surface is dissociated with EROS. Further studies are required to delineate how EROS dissociates from NOX2 during its transport to cell surface, which may be a potential mechanism for regulation of NOX2 activation.
History
DepositionAug 11, 2023Deposition site: PDBJ / Processing site: PDBC
Revision 1.0May 22, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 20, 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 1.2Dec 4, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _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: monoclonal antibody 7G5 heavy chain
D: Cytochrome b-245 chaperone 1
E: monoclonal antibody 7G5 light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)149,16112
Polymers145,0135
Non-polymers4,1477
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Cytochrome b-245 ... , 3 types, 3 molecules ABD

#1: 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: 14663.218 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 / 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: 64853.066 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CYBB, NOX2 / Production host: Homo sapiens (human) / References: UniProt: P04839, Oxidoreductases
#4: Protein Cytochrome b-245 chaperone 1 / Essential for reactive oxygen species protein / Eros


Mass: 18491.500 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CYBC1, C17orf62, EROS / Production host: Homo sapiens (human) / References: UniProt: Q9BQA9

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Antibody , 2 types, 2 molecules CE

#3: Antibody monoclonal antibody 7G5 heavy chain


Mass: 23484.305 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Oryctolagus cuniculus (rabbit) / Production host: Homo sapiens (human)
#5: Antibody monoclonal 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: Homo sapiens (human)

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Sugars , 2 types, 3 molecules

#6: 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.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam 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
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}}LINUCSPDB-CARE
#9: 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.: 1 / Source method: obtained synthetically / Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
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 , 3 types, 4 molecules

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


Mass: 760.076 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C42H82NO8P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
#8: 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
#10: Chemical ChemComp-LBN / 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine / (2R)-2-[(9Z)-9-Octadecenoyloxy]-3-(palmitoyloxy)propyl 2-(trimethylammonio)ethyl phosphate


Mass: 760.076 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C42H82NO8P / Comment: phospholipid*YM

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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: NADPH oxidase 2 in complex with Cytochrome b-245 light chain, Cytochrome b-245 chaperone 1 and a monoclonal antibody
Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
31Oryctolagus cuniculus (rabbit)9986
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD
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: 2500 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 52 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.56 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 131926 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00610471
ELECTRON MICROSCOPYf_angle_d0.94914231
ELECTRON MICROSCOPYf_dihedral_angle_d7.181486
ELECTRON MICROSCOPYf_chiral_restr0.0421598
ELECTRON MICROSCOPYf_plane_restr0.0051745

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