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- EMDB-25836: CryoEM Structure of sFab COP-3 Complex with human claudin-4 and C... -

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Basic information

Entry
Database: EMDB / ID: EMD-25836
TitleCryoEM Structure of sFab COP-3 Complex with human claudin-4 and Clostridium perfringens enterotoxin C-terminal domain focused map
Map data
Sample
  • Complex: Human claudin-4/cCpE/COP-3 Complex
    • Protein or peptide: COP-3 H chain
    • Protein or peptide: COP-3 L chain
    • Protein or peptide: Clostridium perfringens enterotoxin C-terminal domain
KeywordsClaudin / cell adhesion / enterotoxin / Fab / antibody fragment / MEMBRANE PROTEIN
Function / homologyClostridium enterotoxin / Clostridium enterotoxin / toxin activity / extracellular region / Heat-labile enterotoxin B chain
Function and homology information
Biological speciesHomo sapiens (human) / Clostridium perfringens (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsVecchio AJ / Orlando BJ
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM138368 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM117372 United States
CitationJournal: J Biol Chem / Year: 2022
Title: Development, structure, and mechanism of synthetic antibodies that target claudin and Clostridium perfringens enterotoxin complexes.
Authors: Benjamin J Orlando / Pawel K Dominik / Sourav Roy / Chinemerem P Ogbu / Satchal K Erramilli / Anthony A Kossiakoff / Alex J Vecchio /
Abstract: Strains of Clostridium perfringens produce a two-domain enterotoxin (CpE) that afflicts humans and domesticated animals, causing prevalent gastrointestinal illnesses. CpE's C-terminal domain (cCpE) ...Strains of Clostridium perfringens produce a two-domain enterotoxin (CpE) that afflicts humans and domesticated animals, causing prevalent gastrointestinal illnesses. CpE's C-terminal domain (cCpE) binds cell surface receptors, followed by a restructuring of its N-terminal domain to form a membrane-penetrating β-barrel pore, which is toxic to epithelial cells of the gut. The claudin family of membrane proteins are known receptors for CpE and also control the architecture and function of cell-cell contacts (tight junctions) that create barriers to intercellular molecular transport. CpE binding and assembly disables claudin barrier function and induces cytotoxicity via β-pore formation, disrupting gut homeostasis; however, a structural basis of this process and strategies to inhibit the claudin-CpE interactions that trigger it are both lacking. Here, we used a synthetic antigen-binding fragment (sFab) library to discover two sFabs that bind claudin-4 and cCpE complexes. We established these sFabs' mode of molecular recognition and binding properties and determined structures of each sFab bound to claudin-4-cCpE complexes using cryo-EM. The structures reveal that the sFabs bind a shared epitope, but conform distinctly, which explains their unique binding equilibria. Mutagenesis of antigen/sFab interfaces observed therein result in binding changes, validating the structures, and uncovering the sFab's targeting mechanism. From these insights, we generated a model for CpE's claudin-bound β-pore that predicted sFabs would not prevent cytotoxicity, which we then verified in vivo. Taken together, this work demonstrates the development and mechanism of claudin/cCpE-binding sFabs that provide a framework and strategy for obstructing claudin/CpE assembly to treat CpE-linked gastrointestinal diseases.
History
DepositionJan 1, 2022-
Header (metadata) releaseAug 10, 2022-
Map releaseAug 10, 2022-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25836.png

Downloads & links

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Map

FileDownload / File: emd_25836.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.871 Å
Density
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-10.745827 - 10.558854999999999
Average (Standard dev.)0.00062536635 (±0.082892664)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-160-160-160
Dimensions320320320
Spacing320320320
CellA=B=C: 278.72 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_25836_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_25836_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 : Human claudin-4/cCpE/COP-3 Complex

EntireName: Human claudin-4/cCpE/COP-3 Complex
Components
  • Complex: Human claudin-4/cCpE/COP-3 Complex
    • Protein or peptide: COP-3 H chain
    • Protein or peptide: COP-3 L chain
    • Protein or peptide: Clostridium perfringens enterotoxin C-terminal domain

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Supramolecule #1: Human claudin-4/cCpE/COP-3 Complex

SupramoleculeName: Human claudin-4/cCpE/COP-3 Complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: COP-3 sFab fragment bound to Claudin-4/cCpE complex
Molecular weightTheoretical: 85 kDa/nm

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Macromolecule #1: COP-3 H chain

MacromoleculeName: COP-3 H chain / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: EISEVQLVES GGGLVQPGGS LRLSCAASGF NFYSSSIHWV RQAPGKGLEW VAYISSYSGY TYYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARGYGYFDYN FSVGYALDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK D YFPEPVTV ...String:
EISEVQLVES GGGLVQPGGS LRLSCAASGF NFYSSSIHWV RQAPGKGLEW VAYISSYSGY TYYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARGYGYFDYN FSVGYALDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK D YFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDKKVE PKSCDKTHT

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Macromolecule #2: COP-3 L chain

MacromoleculeName: COP-3 L chain / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSHPWYYPI TFGQGTKVEI KRTVAAPSVF IFPPSDSQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS G NSQESVTE ...String:
SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSHPWYYPI TFGQGTKVEI KRTVAAPSVF IFPPSDSQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS G NSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC

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Macromolecule #3: Clostridium perfringens enterotoxin C-terminal domain

MacromoleculeName: Clostridium perfringens enterotoxin C-terminal domain / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Clostridium perfringens (bacteria)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
MSTDIEKEIL DLAAATERLN LTDALNSNPA GNLYDWRSSN SYPWTQKLNL HLTITATGQK YRILASKIVD FNIYSNNFNN LVKLEQSLGD GVKDHYVDIS LDAGQYVLVM KANSSYSGNY PYSILFQKFG LVPR

UniProtKB: Heat-labile enterotoxin B chain

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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

Concentration6.0 mg/mL
BufferpH: 7.4
GridModel: Quantifoil R1.2/1.3 / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Pressure: 0.001 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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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: 2.2 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 120000
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 3758631 / Average electron dose: 39.6 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 305927
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7tdn

Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 305927
FSC plot (resolution estimation)

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