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- PDB-9arl: CryoEM structure of BoNT-NTNH-OrfX2 complex from Clostridium botu... -

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Basic information

Entry
Database: PDB / ID: 9arl
TitleCryoEM structure of BoNT-NTNH-OrfX2 complex from Clostridium botulinum strain A1-ST7B, major class
Components
  • Botulinum neurotoxin type A
  • NtnH
  • OrfX2
KeywordsTOXIN / Botulinum neurotoxin / Progenitor toxin complex (PTC)
Function / homology
Function and homology information


bontoxilysin / host cell presynaptic membrane / host cell cytoplasmic vesicle / host cell cytosol / protein transmembrane transporter activity / metalloendopeptidase activity / toxin activity / host cell plasma membrane / proteolysis / extracellular region ...bontoxilysin / host cell presynaptic membrane / host cell cytoplasmic vesicle / host cell cytosol / protein transmembrane transporter activity / metalloendopeptidase activity / toxin activity / host cell plasma membrane / proteolysis / extracellular region / zinc ion binding / membrane
Similarity search - Function
Clostridium P47 protein / Clostridium P-47-like / Nontoxic nonhaemagglutinin C-terminal / Nontoxic nonhaemagglutinin C-terminal / Botulinum neurotoxin, helical domain / Clostridium neurotoxin, translocation / Clostridium neurotoxin, Translocation domain / Clostridium neurotoxin, translocation domain / Clostridium neurotoxin, receptor-binding C-terminal / Clostridium neurotoxin, C-terminal receptor binding ...Clostridium P47 protein / Clostridium P-47-like / Nontoxic nonhaemagglutinin C-terminal / Nontoxic nonhaemagglutinin C-terminal / Botulinum neurotoxin, helical domain / Clostridium neurotoxin, translocation / Clostridium neurotoxin, Translocation domain / Clostridium neurotoxin, translocation domain / Clostridium neurotoxin, receptor-binding C-terminal / Clostridium neurotoxin, C-terminal receptor binding / Clostridial neurotoxin zinc protease / Botulinum/Tetanus toxin, catalytic chain / Clostridium neurotoxin, receptor binding N-terminal / Clostridium neurotoxin, N-terminal receptor binding / Kunitz inhibitor STI-like superfamily / Neutral zinc metallopeptidases, zinc-binding region signature. / Concanavalin A-like lectin/glucanase domain superfamily
Similarity search - Domain/homology
OrfX2 / NtnH / Botulinum neurotoxin type A
Similarity search - Component
Biological speciesClostridium botulinum A str. Hall (bacteria)
Clostridium botulinum A (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsGao, L.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI139087 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI158503 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R21AI163178 United States
CitationJournal: Nat Struct Mol Biol / Year: 2025
Title: Botulinum neurotoxins exploit host digestive proteases to boost their oral toxicity via activating OrfXs/P47.
Authors: Linfeng Gao / Maria Barbara Nowakowska / Katja Selby / Adina Przykopanski / Baohua Chen / Maren Krüger / François Paul Douillard / Kwok-Ho Lam / Peng Chen / Ting Huang / Nigel Peter Minton ...Authors: Linfeng Gao / Maria Barbara Nowakowska / Katja Selby / Adina Przykopanski / Baohua Chen / Maren Krüger / François Paul Douillard / Kwok-Ho Lam / Peng Chen / Ting Huang / Nigel Peter Minton / Martin Bernhard Dorner / Brigitte Gertrud Dorner / Andreas Rummel / Miia Lindström / Rongsheng Jin /
Abstract: Botulinum neurotoxins (BoNTs) rank among the most potent toxins and many of them are produced by bacteria carrying the orfX gene cluster that also encodes four nontoxic proteins (OrfX1, OrfX2, OrfX3 ...Botulinum neurotoxins (BoNTs) rank among the most potent toxins and many of them are produced by bacteria carrying the orfX gene cluster that also encodes four nontoxic proteins (OrfX1, OrfX2, OrfX3 and P47). The orfX gene cluster is also found in the genomes of many non-BoNT-producing bacteria, often alongside genes encoding oral insecticidal toxins. However, the functions of these OrfXs and P47 remain elusive. Here, we demonstrate that the combined action of all four components (OrfXs and P47) drastically boosts the oral toxicity of BoNT in mice, following proteolytic activation by digestive proteases that oral toxins regularly confront. In particular, OrfX2 adopts a self-inhibiting state, engaging with BoNT through another clostridial protein, nontoxic non-hemagglutinin (NTNH), only after proteolytic activation. Cryo-electron microscopy studies unveil that two molecules of protease-activated OrfX2 simultaneously associate with NTNH, a binding mode crucial for boosting BoNT oral toxicity. Collectively, these studies offer novel insights into the physiological functions and regulatory mechanisms of OrfXs and P47 of BoNTs, shedding light on the pathogenesis of other bacterial toxins associated with homologous OrfXs and P47.
History
DepositionFeb 23, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 22, 2025Provider: repository / Type: Initial release
Revision 1.0Jan 22, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jan 22, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
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Revision 1.1Feb 5, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Botulinum neurotoxin type A
B: NtnH
C: OrfX2


Theoretical massNumber of molelcules
Total (without water)369,5893
Polymers369,5893
Non-polymers00
Water00
1


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

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Components

#1: Protein Botulinum neurotoxin type A / BoNT/A / Bontoxilysin-A / BOTOX


Mass: 149479.859 Da / Num. of mol.: 1 / Mutation: E224Q, R363A, Y366F
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridium botulinum A str. Hall (bacteria)
Gene: botA, bna, CBO0806, CLC_0862 / Production host: Escherichia coli (E. coli) / References: UniProt: P0DPI1
#2: Protein NtnH


Mass: 135285.750 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridium botulinum A (bacteria) / Gene: ntnH / Production host: Escherichia coli (E. coli) / References: UniProt: B0FNQ9
#3: Protein OrfX2


Mass: 84823.297 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridium botulinum A (bacteria) / Production host: Escherichia coli (E. coli) / References: UniProt: B0FNQ5
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: Ternary complex of M-PTC/A1-ST7B with OrfX2/A1-ST7B, major class
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.352 MDa / Experimental value: NO
Source (natural)Organism: Clostridium botulinum A (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 6
Buffer component
IDConc.NameFormulaBuffer-ID
10.2 MMESMES1
20.1 Msodium chlorideNaCl1
SpecimenConc.: 0.5 mg/ml / Embedding 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: 2300 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 3

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.4.1particle selection
9PHENIXmodel refinement
12cryoSPARC4.4.1classification
13cryoSPARC4.4.13D reconstruction
CTF correctionType: NONE
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 135181 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00324560
ELECTRON MICROSCOPYf_angle_d0.46133280
ELECTRON MICROSCOPYf_dihedral_angle_d11.6599023
ELECTRON MICROSCOPYf_chiral_restr0.0423701
ELECTRON MICROSCOPYf_plane_restr0.0034288

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