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- EMDB-43784: CryoEM structure of BoNT-NTNH-OrfX2 complex from Clostridium botu... -

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Basic information

Entry
Database: EMDB / ID: EMD-43784
TitleCryoEM structure of BoNT-NTNH-OrfX2 complex from Clostridium botulinum E1, major class
Map data
Sample
  • Complex: Ternary complex of M-PTC/E with OrfX2, major class
    • Protein or peptide: Toxin
    • Protein or peptide: Botulinum neurotoxin
    • Protein or peptide: Peptidase M27
KeywordsBotulinum neurotoxin / Progenitor toxin complex (PTC) / TOXIN
Function / homology
Function and homology information


protein transmembrane transporter activity / metalloendopeptidase activity / toxin activity / proteolysis / extracellular region / zinc ion binding
Similarity search - Function
Nontoxic nonhaemagglutinin C-terminal / Nontoxic nonhaemagglutinin C-terminal / Botulinum neurotoxin, helical domain / Clostridium P47 protein / Clostridium P-47 protein / Clostridium neurotoxin, translocation / Clostridium neurotoxin, Translocation domain / Clostridium neurotoxin, translocation domain / Clostridial neurotoxin zinc protease / Botulinum/Tetanus toxin, catalytic chain ...Nontoxic nonhaemagglutinin C-terminal / Nontoxic nonhaemagglutinin C-terminal / Botulinum neurotoxin, helical domain / Clostridium P47 protein / Clostridium P-47 protein / Clostridium neurotoxin, translocation / Clostridium neurotoxin, Translocation domain / Clostridium neurotoxin, translocation domain / Clostridial neurotoxin zinc protease / Botulinum/Tetanus toxin, catalytic chain / Clostridium neurotoxin, receptor-binding C-terminal / Clostridium neurotoxin, receptor binding N-terminal / Clostridium neurotoxin, C-terminal receptor binding / Clostridium neurotoxin, N-terminal receptor binding / Kunitz inhibitor STI-like superfamily / Concanavalin A-like lectin/glucanase domain superfamily
Similarity search - Domain/homology
Peptidase M27 / Toxin / Botulinum neurotoxin
Similarity search - Component
Biological speciesClostridium botulinum E1 str. 'BoNT E Beluga' (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsGao L
Funding support United States, 3 items
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, 2024-
Header (metadata) releaseJan 22, 2025-
Map releaseJan 22, 2025-
UpdateMay 28, 2025-
Current statusMay 28, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43784.png

Downloads & links

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Map

FileDownload / File: emd_43784.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.79 Å/pix.
x 512 pix.
= 403.456 Å		0.79 Å/pix.
x 512 pix.
= 403.456 Å		0.79 Å/pix.
x 512 pix.
= 403.456 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.788 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.2508308 - 0.5026714
Average (Standard dev.)0.0000030307906 (±0.008585676)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 403.456 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_43784_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_43784_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of M-PTC/E with OrfX2, major class

EntireName: Ternary complex of M-PTC/E with OrfX2, major class
Components
  • Complex: Ternary complex of M-PTC/E with OrfX2, major class
    • Protein or peptide: Toxin
    • Protein or peptide: Botulinum neurotoxin
    • Protein or peptide: Peptidase M27

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Supramolecule #1: Ternary complex of M-PTC/E with OrfX2, major class

SupramoleculeName: Ternary complex of M-PTC/E with OrfX2, major class / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Clostridium botulinum E1 str. 'BoNT E Beluga' (bacteria)
Molecular weightTheoretical: 346 KDa

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Macromolecule #1: Toxin

MacromoleculeName: Toxin / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Clostridium botulinum E1 str. 'BoNT E Beluga' (bacteria)
Molecular weightTheoretical: 84.690398 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTNLKPYIIY DWKETILKNS KDNYSINESI PKIFSKKICG GRFFNSTLSG NWKSWTLTDE GEGPHPVLKC TIDNGYLEIY SNTSSEKHS LKDIEIKVCM SIKPNSDGTH SLCKNSFYIK TNSLKLSEDR LILSHCLDKL ILAWFKDNHK YIELFINRSR I QTRVEGDL ...String:
MTNLKPYIIY DWKETILKNS KDNYSINESI PKIFSKKICG GRFFNSTLSG NWKSWTLTDE GEGPHPVLKC TIDNGYLEIY SNTSSEKHS LKDIEIKVCM SIKPNSDGTH SLCKNSFYIK TNSLKLSEDR LILSHCLDKL ILAWFKDNHK YIELFINRSR I QTRVEGDL SLLGWDIESS VSYKTMNEFI KKDNLYEKKF HQYMEVRRNE YTIDGEFGPW QMTTGADGQN IRFLCPIKSA TY KINDDVY IAKPDNFIII QVDLKYFDSK TTIIDPSGLN NGQQFNLKVK TDSTDEINAV ILVGSRITDV NEDLYPGDDV SLE IVFKTW FNANIQKFTQ IFSYILLNET SKIPEYQWLK PTQISYGSAS VTMPDPSNPN KELSNLDAST FAAMAMVENH KNDR PNHAV DNRFLELSKT PAAFAISMPE FLKHFLVTGL QAMQIDNLDA FEVSSENLVI TNKKKINFGK IQDQNRQVDA LIEPN NFKL AIQNNQVVVE IVDATWQQVV GVTGHFGYRQ AYNLILKNEN NVYKPMLEES GDVTISYMVT EEAWKTTQDA IISATV GLV VGTIIGTAFS KLSDKLYKFL KSKFIVKNKK ASLKISGKDI NEVIEMSDIS KPQLLSIKKA NAKISTEEVG LISQNGS TS LENLAIFKNK PRPIGERVQI LGLKLVSGLI TTFGWSIGFV LPDILKDVIN ANINNNFEVL PGIQQFTQQC IGSIQWPD N SELKIDFAKL QGVYLLGGNL VKIPESN

UniProtKB: Toxin

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Macromolecule #2: Botulinum neurotoxin

MacromoleculeName: Botulinum neurotoxin / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Clostridium botulinum E1 str. 'BoNT E Beluga' (bacteria)
Molecular weightTheoretical: 143.587938 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MPKINSFNYN DPVNDRTILY IKPGGCQEFY KSFNIMKNIW IIPERNVIGT TPQDFHPPTS LKNGDSSYYD PNYLQSDEEK DRFLKIVTK IFNRINNNLS GGILLEELSK ANPYLGNDNT PDNQFHIGDA SAVEIKFSNG SQDILLPNVI IMGAEPDLFE T NSSNISLR ...String:
MPKINSFNYN DPVNDRTILY IKPGGCQEFY KSFNIMKNIW IIPERNVIGT TPQDFHPPTS LKNGDSSYYD PNYLQSDEEK DRFLKIVTK IFNRINNNLS GGILLEELSK ANPYLGNDNT PDNQFHIGDA SAVEIKFSNG SQDILLPNVI IMGAEPDLFE T NSSNISLR NNYMPSNHGF GSIAIVTFSP EYSFRFNDNS MNEFIQDPAL TLMAALIASL HGLYGAKGIT TKYTITQKQN PL ITNIRGT NIEEFLTFGG TDLNIITSAQ SNDIYTNLLA DYKKIASKLS KVQVSNPLLN PYKDVFEAKY GLDKDASGIY SVN INKFND IFKKLYSFTE FDLATKFQVK CRQTYIGQYK YFKLSNLLND SIYNISEGYN INNLKVNFRG QNANLNPRII TPIT GRGLV KKIIRFCKNI VSVKGIRKSI CIEINNGELF FVASENSYND DNINTPKEID DTVTSNNNYE NDLDQVILNF NSESA PGLS DEKLNLTIQN DAYIPKYDSN GTSDIEQHDV NELNVFFYLD AQKVPEGENN VNLTSSIDTA LLEQPKIYTF FSSEFI NNV NKPVQAALFV SWIQQVLVDF TTEANQKSTV DKIADISIVV PYIGLALNIG NEAQKGNFKD ALELLGAGIL LEFEPEL LI PTILVFTIKS FLGSSDNKNK VIKAINNALK ERDEKWKEVY SFIVSNWMTK INTQFNKRKE QMYQALQNQV NAIKTIIE S KYNSYTLEEK NELTNKYDIK QIENELNQKV SIAMNNIDRF LTESSISYLM KLINEVKINK LREYDENVKT YLLNYIIQH GSILGESQQE LNSMVTDTLN NSIPFKLSSY TDDKILISYF NKFFKRIKSS SVLNMRYKND KYVDTSGYDS NININGDVYK YPTNKNQFG IYNDKLSEVN ISQNDYIIYD NKYKNFSISF WVRIPNYDNK IVNVNNEYTI INCMRDNNSG WKVSLNHNEI I WTLQDNAG INQKLAFNYG NANGISDYIN KWIFVTITND RLGDSKLYIN GNLIDQKSIL NLGNIHVSDN ILFKIVNCSY TR YIGIRYF NIFDKELDET EIQTLYSNEP NTNILKDFWG NYLLYDKEYY LLNVLKPNNF IDRRKDSTLS INNIRSTILL ANR LYSGIK VKIQRVNNSS TNDNLVRKND QVYINFVASK THLFPLYADT ATTNKEKTIK ISSSGNRFNQ VVVMNSVGNN CTMN FKNNN GNNIGLLGFK ADTVVASTWY YTHMRDHTNS NGCFWNFISE EHGWQEK

UniProtKB: Botulinum neurotoxin

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Macromolecule #3: Peptidase M27

MacromoleculeName: Peptidase M27 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Clostridium botulinum E1 str. 'BoNT E Beluga' (bacteria)
Molecular weightTheoretical: 136.967375 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKINGNLNID SPVDNKNVAI VRSRKSDVFF KAFQVAPNIW IVPERYYGES LKINEDQKFD GGIYDSNFLS TNNEKDDFLQ ATIKLLQRI NNNVVGAKLL SLISTAIPFP YENNTEDYRQ TNYLSSKNNE HYYTANLVIF GPGSNIIKNN VIYYKKEYAE S GMGTMLEI ...String:
MKINGNLNID SPVDNKNVAI VRSRKSDVFF KAFQVAPNIW IVPERYYGES LKINEDQKFD GGIYDSNFLS TNNEKDDFLQ ATIKLLQRI NNNVVGAKLL SLISTAIPFP YENNTEDYRQ TNYLSSKNNE HYYTANLVIF GPGSNIIKNN VIYYKKEYAE S GMGTMLEI WFQPFLTHKY DEFYVDPALE LIKCLIKSLY YLYGIKPNDN LNIPYRLRNE FNSLEYSELD MIDFLISGGI DY KLLNTNP YWFIDKYFID TSKNFEKYKN DYEIKIKNNN YIANSIKLYL EQKFKINVKD IWELNLSYFS KEFQIMMPER YNN ALNHYY RKEYYVIDYF KNYNINGFKN GQIKTKLPLS KYNKEIINKP ELIVNLINQN NTVLMKSNIY GDGLKGTVDN FYSN YIIPY NLNYEHSINY SYLDNVNIEE IEKIPPINDE DIYPYRKNAD TFIPVYNITK AKEINTTTPL PVNYLQAQMI DSNDI NLSS DFLKVISSKG SLVYSFLNNT MDYLEFIKYD KPIDTDKKYY KWLKAIFRNY SLDITETQEI SNQFGDTKII PWIGRA LNI LNTNNSFVEE FKNLGPISLI NKKENITIPK IKIDEIPSSM LNFSFKDLSE NLFNIYCKNN FYLKKIYYNF LDQWWTQ YY SQYFDLICMA SKSVLAQEKL IKKLIQKQLR YLMENSNISS TNLILINLTT TNTLRDISNQ SQIAINNIDK FFNNAAMC V FENNIYPKFT SFMEQCIKNI NKSTKEFILK CTNINETEKS HLIMQNSFSN LDFDFLDIQN MKNLFNSYTE LLIKEQTSP YELSLYAFQE QDNNVIGDTS GKNTLVEYPK DIGLVYGINN NAIHLTGANQ NIKFTNDYFE NGLTNNFSIY FWLRNLKQNT IKSKLIGSK EDNCGWEIYF ENDGLVFNII DSNGNEKNIY LSNISNNSWH YIVISINRLK DQLLIFIDNI LVANEDIKEI L NIYSSDII SLLSDNNNVY IEGLSVLNKT INSNEILTDY FSDLNNSYIR NFDEEILQYN RTYELFNYVF PEIAINKIEQ NN NIYLSIN NENNLNFKPL KFKLLNTNPN KQYVQKWDEV IFSVLDGTEK YLDISTTNNR IQLVDNKNNA QIFIINNDIF ISN CLTLTY NNVNIYLSIK NQDYNWVICD LNHDIPKKSY LWILKNI

UniProtKB: Peptidase M27

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 6
Component:
ConcentrationFormulaName
0.2 MMESMES
0.1 MNaClsodium chloride
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 4 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.3000000000000003 µm / Nominal defocus min: 0.7000000000000001 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: NONE
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4.1) / Number images used: 384801
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE

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