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- EMDB-26563: Stx2a and DARPin complex -

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Basic information

Entry
Database: EMDB / ID: EMD-26563
TitleStx2a and DARPin complex
Map data
Sample
  • Complex: Ternary complex of stx2 and DARPin
    • Complex: Shiga-like toxin 2 subunit AShiga toxin
      • Complex: Shiga-like toxin 2 subunit BShiga toxin
        • Complex: DARPin
    • Protein or peptide: DARPin
    • Protein or peptide: Shiga-like toxin 2 subunit AShiga toxin
    • Protein or peptide: Shiga-like toxin 2 subunit BShiga toxin
  • Ligand: 3-PYRIDINIUM-1-YLPROPANE-1-SULFONATE
  • Ligand: FORMIC ACID
  • Ligand: SODIUM IONSodium
  • Ligand: 1,2-ETHANEDIOLEthylene glycol
  • Ligand: water
Function / homology
Function and homology information


modulation of host virulence by virus / hemolysis by symbiont of host erythrocytes / rRNA N-glycosylase / rRNA N-glycosylase activity / toxin activity / negative regulation of translation / extracellular region
Similarity search - Function
Shiga-like toxin, subunit A / Shiga-like toxin, beta subunit / Shiga-like toxin beta subunit / Enterotoxin / Ribosome-inactivating protein conserved site / Shiga/ricin ribosomal inactivating toxins active site signature. / Ribosome-inactivating protein / Ribosome-inactivating protein, subdomain 1 / Ribosome-inactivating protein, subdomain 2 / Ribosome-inactivating protein superfamily / Ribosome inactivating protein
Similarity search - Domain/homology
Shiga-like toxin 2 subunit A / Shiga-like toxin 2 subunit B
Similarity search - Component
Biological speciesEscherichia phage 933W (virus) / Synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.5 Å
AuthorsJiang M / Zhang J
Funding support United States, 1 items
OrganizationGrant numberCountry
Welch FoundationA1863 United States
CitationJournal: Bioengineering (Basel) / Year: 2022
Title: A Multi-Specific DARPin Potently Neutralizes Shiga Toxin 2 via Simultaneous Modulation of Both Toxin Subunits.
Authors: Yu Zeng / Mengqiu Jiang / Sally Robinson / Zeyu Peng / Vikas Chonira / Rudo Simeon / Saul Tzipori / Junjie Zhang / Zhilei Chen /
Abstract: Shiga toxin-producing (STEC) is a common cause of bloody diarrhea. The pathology of STEC infection derives from two exotoxins-Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2)-that are secreted by STEC ...Shiga toxin-producing (STEC) is a common cause of bloody diarrhea. The pathology of STEC infection derives from two exotoxins-Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2)-that are secreted by STEC in the gut, from where they are systemically absorbed, causing severe kidney damage leading to hemolytic uremic syndrome (HUS). Currently, there is no effective treatment for HUS, and only supportive care is recommended. We report the engineering of a panel of designed ankyrin repeat proteins (DARPin) with potent neutralization activity against Stx2a, the major subtype associated with HUS. The best dimeric DARPin, SD5, created via a combination of directed evolution and rational design, neutralizes Stx2a with a half maximal effective concentration (EC) of 0.61 nM The two monomeric DARPin constituents of SD5 exhibit complementary functions-SHT targets the enzymatic A subunit of Stx2a and inhibits the toxin's catalytic activity, while DARPin #3 binds the B subunit, based on the cryo-EM study, and induces a novel conformational change in the B subunit that distorts its five-fold symmetry and presumably interferes with toxin attachment to target cells. SD5 was fused to an albumin-binding DARPin, and the resulting trimeric DARPin DA1-SD5 efficiently protects mice in a toxin challenge model, pointing to a high potential of this DARPin as a therapeutic for STEC infection. Finally, the unprecedented toxin conformational change induced by DARPin #3 represents a novel mode of action for neutralizing Stx2 toxicity and reveals new targets for future drug development.
History
DepositionMar 30, 2022-
Header (metadata) releaseApr 12, 2023-
Map releaseApr 12, 2023-
UpdateApr 19, 2023-
Current statusApr 19, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26563.png

Downloads & links

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Map

FileDownload / File: emd_26563.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.73
Minimum - Maximum-1.5965651 - 2.1085691
Average (Standard dev.)0.00063655165 (±0.08646689)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 273.92 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_26563_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_26563_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of stx2 and DARPin

EntireName: Ternary complex of stx2 and DARPin
Components
  • Complex: Ternary complex of stx2 and DARPin
    • Complex: Shiga-like toxin 2 subunit AShiga toxin
      • Complex: Shiga-like toxin 2 subunit BShiga toxin
        • Complex: DARPin
    • Protein or peptide: DARPin
    • Protein or peptide: Shiga-like toxin 2 subunit AShiga toxin
    • Protein or peptide: Shiga-like toxin 2 subunit BShiga toxin
  • Ligand: 3-PYRIDINIUM-1-YLPROPANE-1-SULFONATE
  • Ligand: FORMIC ACID
  • Ligand: SODIUM IONSodium
  • Ligand: 1,2-ETHANEDIOLEthylene glycol
  • Ligand: water

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Supramolecule #1: Ternary complex of stx2 and DARPin

SupramoleculeName: Ternary complex of stx2 and DARPin / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Escherichia phage 933W (virus)

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Supramolecule #2: Shiga-like toxin 2 subunit A

SupramoleculeName: Shiga-like toxin 2 subunit A / type: complex / ID: 2 / Chimera: Yes / Parent: 1

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Supramolecule #3: Shiga-like toxin 2 subunit B

SupramoleculeName: Shiga-like toxin 2 subunit B / type: complex / ID: 3 / Chimera: Yes / Parent: 2

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Supramolecule #4: DARPin

SupramoleculeName: DARPin / type: complex / ID: 4 / Chimera: Yes / Parent: 3

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Macromolecule #1: DARPin

MacromoleculeName: DARPin / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Synthetic construct (others)
Molecular weightTheoretical: 19.86341 KDa
Recombinant expressionOrganism: Escherichia coli-Pichia pastoris shuttle vector pPpARG4 (others)
SequenceString:
GKKLLEAARA GQDDEVRILM ANGADVNACD SNGRTPLHLA AVRGHLEIVE VLLKNGADVN AWDVIGKTPL HLAADGGHLE IVEVLLKNG ADVNAWDLIG RTPLHLAALD GHLEIVEVLL EHGADVNAYD RPGHTPLHLA ARRGHLEIVE VLLKYGADVN A QDKFGKTA FDISIDNGNE DLAEILQ

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Macromolecule #2: Shiga-like toxin 2 subunit A

MacromoleculeName: Shiga-like toxin 2 subunit A / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: rRNA N-glycosylase
Source (natural)Organism: Escherichia phage 933W (virus)
Molecular weightTheoretical: 33.228215 KDa
Recombinant expressionOrganism: Escherichia coli-Pichia pastoris shuttle vector pPpARG4 (others)
SequenceString: REFTIDFSTQ QSYVSSLNSI RTEISTPLEH ISQGTTSVSV INHTPPGSYF AVDIRGLDVY QARFDHLRLI IEQNNLYVAG FVNTATNTF YRFSDFTHIS VPGVTTVSMT TDSSYTTLQR VAALERSGMQ ISRHSLVSSY LALMEFSGNT MTRDASRAVL R FVTVTAEA ...String:
REFTIDFSTQ QSYVSSLNSI RTEISTPLEH ISQGTTSVSV INHTPPGSYF AVDIRGLDVY QARFDHLRLI IEQNNLYVAG FVNTATNTF YRFSDFTHIS VPGVTTVSMT TDSSYTTLQR VAALERSGMQ ISRHSLVSSY LALMEFSGNT MTRDASRAVL R FVTVTAEA LRFRQIQREF RQALSETAPV YTMTPGDVDL TLNWGRISNV LPEYRGEDGV RVGRISFNNI SAILGTVAVI LN CHHQGAR SVRAVNEESQ PECQITGDRP VIKINNTLWE SNTAAAFLNR KSQFLYTTGK

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Macromolecule #3: Shiga-like toxin 2 subunit B

MacromoleculeName: Shiga-like toxin 2 subunit B / type: protein_or_peptide / ID: 3 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Escherichia phage 933W (virus)
Molecular weightTheoretical: 7.82459 KDa
Recombinant expressionOrganism: Escherichia coli-Pichia pastoris shuttle vector pPpARG4 (others)
SequenceString:
ADCAKGKIEF SKYNEDDTFT VKVDGKEYWT SRWNLQPLLQ SAQLTGMTVT IKSSTCESGS GFAEVQFNND

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Macromolecule #4: 3-PYRIDINIUM-1-YLPROPANE-1-SULFONATE

MacromoleculeName: 3-PYRIDINIUM-1-YLPROPANE-1-SULFONATE / type: ligand / ID: 4 / Number of copies: 4 / Formula: 1PS
Molecular weightTheoretical: 201.243 Da
Chemical component information

ChemComp-1PS:
3-PYRIDINIUM-1-YLPROPANE-1-SULFONATE

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Macromolecule #5: FORMIC ACID

MacromoleculeName: FORMIC ACID / type: ligand / ID: 5 / Number of copies: 15 / Formula: FMT
Molecular weightTheoretical: 46.025 Da
Chemical component information

ChemComp-FMT:
FORMIC ACID / Formic acid

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Macromolecule #6: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 6 / Number of copies: 5
Molecular weightTheoretical: 22.99 Da

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Macromolecule #7: 1,2-ETHANEDIOL

MacromoleculeName: 1,2-ETHANEDIOL / type: ligand / ID: 7 / Number of copies: 5 / Formula: EDO
Molecular weightTheoretical: 62.068 Da
Chemical component information

ChemComp-EDO:
1,2-ETHANEDIOL / Ethylene glycol

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Macromolecule #8: water

MacromoleculeName: water / type: ligand / ID: 8 / Number of copies: 511 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1r4p

Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 108744

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