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- PDB-6wxv: CryoEM structure of mouse DUOX1-DUOXA1 complex in the presence of... -

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Basic information

Entry
Database: PDB / ID: 6wxv
TitleCryoEM structure of mouse DUOX1-DUOXA1 complex in the presence of NADPH
Components(Dual oxidase ...) x 2
KeywordsMEMBRANE PROTEIN / protein complex / NADPH oxidase / ROS production
Function / homology
Function and homology information


regulation of thyroid hormone generation / Thyroxine biosynthesis / NAD(P)H oxidase (H2O2-forming) / cuticle development / positive regulation of hydrogen peroxide biosynthetic process / superoxide-generating NAD(P)H oxidase activity / hormone biosynthetic process / NAD(P)H oxidase H2O2-forming activity / NADPH oxidase complex / thyroid hormone generation ...regulation of thyroid hormone generation / Thyroxine biosynthesis / NAD(P)H oxidase (H2O2-forming) / cuticle development / positive regulation of hydrogen peroxide biosynthetic process / superoxide-generating NAD(P)H oxidase activity / hormone biosynthetic process / NAD(P)H oxidase H2O2-forming activity / NADPH oxidase complex / thyroid hormone generation / superoxide anion generation / hydrogen peroxide biosynthetic process / positive regulation of cell motility / hydrogen peroxide metabolic process / positive regulation of wound healing / cell leading edge / response to cAMP / positive regulation of neuron differentiation / reactive oxygen species metabolic process / peroxidase activity / cytokine-mediated signaling pathway / defense response / positive regulation of reactive oxygen species metabolic process / protein transport / regulation of inflammatory response / response to oxidative stress / apical plasma membrane / heme binding / calcium ion binding / endoplasmic reticulum membrane / cell surface / endoplasmic reticulum / membrane / plasma membrane
Similarity search - Function
Dual oxidase maturation factor / Dual oxidase, peroxidase domain / Dual oxidase maturation factor / 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 ...Dual oxidase maturation factor / Dual oxidase, peroxidase domain / Dual oxidase maturation factor / 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 / Haem peroxidase, animal-type / Haem peroxidase domain superfamily, animal type / Animal haem peroxidase / Animal heme peroxidase superfamily profile. / EF hand / Haem peroxidase superfamily / 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 / EF-hand domain pair / EF-hand, calcium binding motif / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain. / EF-hand calcium-binding domain profile. / EF-hand domain / EF-hand domain pair
Similarity search - Domain/homology
FLAVIN-ADENINE DINUCLEOTIDE / HEME B/C / Chem-NDP / DIUNDECYL PHOSPHATIDYL CHOLINE / NAD(P)H oxidase (H2O2-forming) / Dual oxidase maturation factor 1
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsSun, J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL143037 United States
CitationJournal: Nat Struct Mol Biol / Year: 2020
Title: Structures of mouse DUOX1-DUOXA1 provide mechanistic insights into enzyme activation and regulation.
Authors: Ji Sun /
Abstract: DUOX1, an NADPH oxidase family member, catalyzes the production of hydrogen peroxide. DUOX1 is expressed in various tissues, including the thyroid and respiratory tract, and plays a crucial role in ...DUOX1, an NADPH oxidase family member, catalyzes the production of hydrogen peroxide. DUOX1 is expressed in various tissues, including the thyroid and respiratory tract, and plays a crucial role in processes such as thyroid hormone biosynthesis and innate host defense. DUOX1 co-assembles with its maturation factor DUOXA1 to form an active enzyme complex. However, the molecular mechanisms for activation and regulation of DUOX1 remain mostly unclear. Here, I present cryo-EM structures of the mammalian DUOX1-DUOXA1 complex, in the absence and presence of substrate NADPH, as well as DUOX1-DUOXA1 in an unexpected dimer-of-dimers configuration. These structures reveal atomic details of the DUOX1-DUOXA1 interaction, a lipid-mediated NADPH-binding pocket and the electron transfer path. Furthermore, biochemical and structural analyses indicate that the dimer-of-dimers configuration represents an inactive state of DUOX1-DUOXA1, suggesting an oligomerization-dependent regulatory mechanism. Together, my work provides structural bases for DUOX1-DUOXA1 activation and regulation.
History
DepositionMay 12, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 2, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 17, 2021Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _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

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Structure visualization

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Assembly

Deposited unit
A: Dual oxidase 1
B: Dual oxidase maturation factor 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)218,58613
Polymers213,3602
Non-polymers5,22711
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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Dual oxidase ... , 2 types, 2 molecules AB

#1: Protein Dual oxidase 1


Mass: 175739.812 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Duox1 / Production host: Homo sapiens (human) / References: UniProt: A2AQ92
#2: Protein Dual oxidase maturation factor 1 / Dual oxidase activator 1


Mass: 37619.738 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Duoxa1 / Production host: Homo sapiens (human) / References: UniProt: Q8VE49

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Sugars , 2 types, 5 molecules

#3: Polysaccharide alpha-D-mannopyranose-(1-3)-alpha-D-mannopyranose-(1-6)-beta-D-mannopyranose-(1-4)-2-acetamido-2- ...alpha-D-mannopyranose-(1-3)-alpha-D-mannopyranose-(1-6)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 910.823 Da / Num. of mol.: 1 / Source method: obtained synthetically
DescriptorTypeProgram
DManpa1-3DManpa1-6DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/3,5,4/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3/a4-b1_b4-c1_c6-d1_d3-e1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(6+1)][a-D-Manp]{[(3+1)][a-D-Manp]{}}}}}}LINUCSPDB-CARE
#5: 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.: 4 / 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 , 5 types, 6 molecules

#4: Chemical ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE


Mass: 785.550 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: C27H33N9O15P2 / Comment: FAD*YM
#6: Chemical ChemComp-HEB / HEME B/C / HYBRID BETWEEN B AND C TYPE HEMES (PROTOPORPHYRIN IX CONTAINING FE)


Mass: 618.503 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C34H34FeN4O4
#7: Chemical ChemComp-NDP / NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE


Mass: 745.421 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: C21H30N7O17P3 / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical ChemComp-PLC / DIUNDECYL PHOSPHATIDYL CHOLINE


Mass: 622.834 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: C32H65NO8P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
#9: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca

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Details

Has ligand of interestY

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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: mouse DUOX1-DUOXA1 complex with NADPH / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.22 MDa / Experimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 65.4 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM softwareName: cryoSPARC / Version: 2.13 / Category: 3D reconstruction
CTF correctionType: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 667038 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00410933
ELECTRON MICROSCOPYf_angle_d0.70214926
ELECTRON MICROSCOPYf_dihedral_angle_d24.9431655
ELECTRON MICROSCOPYf_chiral_restr0.0431643
ELECTRON MICROSCOPYf_plane_restr0.0051857

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