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- PDB-7tdn: CryoEM Structure of sFab COP-3 Complex with human claudin-4 and C... -

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Basic information

Entry
Database: PDB / ID: 7tdn
TitleCryoEM Structure of sFab COP-3 Complex with human claudin-4 and Clostridium perfringens enterotoxin C-terminal domain
Components
  • COP-3 Fab Heavy chain
  • COP-3 Fab Light chain
  • Claudin-4CLDN4
  • Heat-labile enterotoxin B chain
KeywordsMEMBRANE PROTEIN / Claudin / cell adhesion / enterotoxin / Fab / antibody fragment
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5 Å
AuthorsVecchio, A.J.
Funding support United States, 2items
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, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 9, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 24, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Sep 21, 2022Group: Database references / Category: citation / Item: _citation.journal_volume
Revision 1.3Jan 17, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Claudin-4
B: Heat-labile enterotoxin B chain
H: COP-3 Fab Heavy chain
L: COP-3 Fab Light chain


Theoretical massNumber of molelcules
Total (without water)86,2214
Polymers86,2214
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Claudin-4 / CLDN4 / Clostridium perfringens enterotoxin receptor / CPE-R / CPE-receptor / Williams-Beuren syndrome ...Clostridium perfringens enterotoxin receptor / CPE-R / CPE-receptor / Williams-Beuren syndrome chromosomal region 8 protein


Mass: 22090.201 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CLDN4, CPER, CPETR1, WBSCR8 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: O14493
#2: Protein Heat-labile enterotoxin B chain


Mass: 15114.945 Da / Num. of mol.: 1 / Fragment: C-terminal domain (UNP residues 192-319)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridium perfringens (bacteria) / Gene: cpe / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P01558
#3: Antibody COP-3 Fab Heavy chain


Mass: 25286.066 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#4: Antibody COP-3 Fab Light chain


Mass: 23729.328 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)

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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: Human claudin-4/cCpE/COP-3 Complex / Type: COMPLEX
Details: COP-3 sFab fragment bound to Claudin-4/cCpE complex
Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 85 kDa/nm / Experimental value: NO
Buffer solutionpH: 7.4
SpecimenConc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 120000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 39.6 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3758631

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
2cryoSPARCimage acquisition
4cryoSPARCCTF correction
7Coot0.9model fitting
12cryoSPARC3D reconstruction
13PHENIX1.19.2-4158model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 305927
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 305927 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 413.26 / Protocol: FLEXIBLE FIT / Space: REAL
Atomic model building

3D fitting-ID: 1 / Accession code: 7TDM / Initial refinement model-ID: 1 / PDB-ID: 7TDM

7tdm

/ Source name: PDB / Type: experimental model

IDPdb chain-ID
1A
2B
3H
4L
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0025800
ELECTRON MICROSCOPYf_angle_d0.6267901
ELECTRON MICROSCOPYf_dihedral_angle_d5.725804
ELECTRON MICROSCOPYf_chiral_restr0.043904
ELECTRON MICROSCOPYf_plane_restr0.004998

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