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- EMDB-25835: CryoEM Structure of sFab COP-3 Complex with human claudin-4 and C... -

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Basic information

Entry
Database: EMDB / ID: EMD-25835
TitleCryoEM Structure of sFab COP-3 Complex with human claudin-4 and Clostridium perfringens enterotoxin C-terminal domain
Map data
Sample
  • Complex: Human claudin-4/cCpE/COP-3 Complex
    • Protein or peptide: Claudin-4CLDN4
    • Protein or peptide: Heat-labile enterotoxin B chain
    • Protein or peptide: COP-3 Fab Heavy chain
    • Protein or peptide: COP-3 Fab Light chain
KeywordsClaudin / cell adhesion / enterotoxin / Fab / antibody fragment / MEMBRANE PROTEIN
Function / homology
Function and homology information


positive regulation of metallopeptidase activity / calcium-independent cell-cell adhesion via plasma membrane cell-adhesion molecules / Tight junction interactions / apicolateral plasma membrane / bicellular tight junction assembly / regulation of cell morphogenesis / tight junction / chloride channel activity / positive regulation of wound healing / renal absorption ...positive regulation of metallopeptidase activity / calcium-independent cell-cell adhesion via plasma membrane cell-adhesion molecules / Tight junction interactions / apicolateral plasma membrane / bicellular tight junction assembly / regulation of cell morphogenesis / tight junction / chloride channel activity / positive regulation of wound healing / renal absorption / chloride channel complex / lateral plasma membrane / bicellular tight junction / establishment of skin barrier / basal plasma membrane / response to progesterone / female pregnancy / circadian rhythm / cell-cell junction / transmembrane signaling receptor activity / toxin activity / cell adhesion / positive regulation of cell migration / apical plasma membrane / structural molecule activity / extracellular region / identical protein binding / plasma membrane
Similarity search - Function
Claudin-4 / Claudin / Claudin, conserved site / Claudin family signature. / Clostridium enterotoxin / Clostridium enterotoxin / PMP-22/EMP/MP20/Claudin family / PMP-22/EMP/MP20/Claudin superfamily
Similarity search - Domain/homology
Claudin-4 / Heat-labile enterotoxin B chain
Similarity search - Component
Biological speciesHomo sapiens (human) / Clostridium perfringens (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.0 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) releaseFeb 9, 2022-
Map releaseFeb 9, 2022-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7tdn
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7tdn
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_25835.png

Downloads & links

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Map

FileDownload / File: emd_25835.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 / Movie #1: 0.2
Minimum - Maximum-0.742016 - 1.221011
Average (Standard dev.)0.0006277186 (±0.04350493)
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 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8710.8710.871
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z278.720278.720278.720
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ256256256
MAP C/R/S123
start NC/NR/NS-160-160-160
NC/NR/NS320320320
D min/max/mean-0.7421.2210.001

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Supplemental data

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Half map: #2

Fileemd_25835_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_25835_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: Claudin-4CLDN4
    • Protein or peptide: Heat-labile enterotoxin B chain
    • Protein or peptide: COP-3 Fab Heavy chain
    • Protein or peptide: COP-3 Fab Light chain

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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: Claudin-4

MacromoleculeName: Claudin-4 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 22.090201 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MASMGLQVMG IALAVLGWLA VMLCCALPMW RVTAFIGSNI VTSQTIWEGL WMNCVVQSTG QMQCKVYDSL LALPQDLQAA RALVIISII VAALGVLLSV VGGKCTNCLE DESAKAKTMI VAGVVFLLAG LMVIVPVSWT AHNIIQDFYN PLVASGQKRE M GASLYVGW ...String:
MASMGLQVMG IALAVLGWLA VMLCCALPMW RVTAFIGSNI VTSQTIWEGL WMNCVVQSTG QMQCKVYDSL LALPQDLQAA RALVIISII VAALGVLLSV VGGKCTNCLE DESAKAKTMI VAGVVFLLAG LMVIVPVSWT AHNIIQDFYN PLVASGQKRE M GASLYVGW AASGLLLLGG GLLCCNCPPR TDKPYSAKYS AARSAAASNY V

UniProtKB: Claudin-4

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Macromolecule #2: Heat-labile enterotoxin B chain

MacromoleculeName: Heat-labile enterotoxin B chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Clostridium perfringens (bacteria)
Molecular weightTheoretical: 15.114945 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
MSTDIEKEIL DLAAATERLN LTDALNSNPA GNLYDWRSSN SYPWTQKLNL HLTITATGQK YRILASKIVD FNIYSNNFNN LVKLEQSLG DGVKDHYVDI SLDAGQYVLV MKANSSYSGN YPYSILFQKF GLVPR

UniProtKB: Heat-labile enterotoxin B chain

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Macromolecule #3: COP-3 Fab Heavy chain

MacromoleculeName: COP-3 Fab Heavy chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.286066 KDa
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 #4: COP-3 Fab Light chain

MacromoleculeName: COP-3 Fab Light chain / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.729328 KDa
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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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:

7tdm

Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 5.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 305927
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

7tdm

chain_id: A, source_name: PDB, initial_model_type: experimental model

7tdm

chain_id: B, source_name: PDB, initial_model_type: experimental model

7tdm

chain_id: H, source_name: PDB, initial_model_type: experimental model

7tdm

chain_id: L, source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 413.26
Output model

PDB-7tdn:
CryoEM Structure of sFab COP-3 Complex with human claudin-4 and Clostridium perfringens enterotoxin C-terminal domain

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