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- PDB-8u5b: Cryo-EM structure of human claudin-4 complex with Clostridium per... -

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Basic information

Entry
Database: PDB / ID: 8u5b
TitleCryo-EM structure of human claudin-4 complex with Clostridium perfringens enterotoxin C-terminal domain and sFab COP-1
Components
  • COP-1 sFab Heavy Chain
  • COP-1 sFab Light Chain
  • Claudin-4
  • Heat-labile enterotoxin B chain
KeywordsMEMBRANE PROTEIN / Claudin / Fab / Toxin
Function / homology
Function and homology information


paracellular transport / calcium-independent cell-cell adhesion via plasma membrane cell-adhesion molecules / Tight junction interactions / bicellular tight junction assembly / apicolateral plasma membrane / regulation of cell morphogenesis / tight junction / positive regulation of wound healing / renal absorption / chloride channel activity ...paracellular transport / calcium-independent cell-cell adhesion via plasma membrane cell-adhesion molecules / Tight junction interactions / bicellular tight junction assembly / apicolateral plasma membrane / regulation of cell morphogenesis / tight junction / positive regulation of wound healing / renal absorption / chloride channel activity / chloride channel complex / bicellular tight junction / lateral plasma membrane / establishment of skin barrier / response to progesterone / basal plasma membrane / female pregnancy / circadian rhythm / transmembrane signaling receptor activity / cell-cell junction / 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
Lauryl Maltose Neopentyl Glycol / Claudin-4 / Heat-labile enterotoxin B chain
Similarity search - Component
Biological speciesHomo sapiens (human)
Clostridium perfringens (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.3 Å
AuthorsVecchio, A.J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM138368 United States
Citation
Journal: Commun Biol / Year: 2024
Title: Structural and biophysical insights into targeting of claudin-4 by a synthetic antibody fragment.
Authors: Satchal K Erramilli / Pawel K Dominik / Chinemerem P Ogbu / Anthony A Kossiakoff / Alex J Vecchio /
Abstract: Claudins are a 27-member family of ~25 kDa membrane proteins that integrate into tight junctions to form molecular barriers at the paracellular spaces between endothelial and epithelial cells. As ...Claudins are a 27-member family of ~25 kDa membrane proteins that integrate into tight junctions to form molecular barriers at the paracellular spaces between endothelial and epithelial cells. As the backbone of tight junction structure and function, claudins are attractive targets for modulating tissue permeability to deliver drugs or treat disease. However, structures of claudins are limited due to their small sizes and physicochemical properties-these traits also make therapy development a challenge. Here we report the development of a synthetic antibody fragment (sFab) that binds human claudin-4 and the determination of a high-resolution structure of it bound to claudin-4/enterotoxin complexes using cryogenic electron microscopy. Structural and biophysical results reveal this sFabs mechanism of select binding to human claudin-4 over other homologous claudins and establish the ability of sFabs to bind hard-to-target claudins to probe tight junction structure and function. The findings provide a framework for tight junction modulation by sFabs for tissue-selective therapies.
#1: Journal: bioRxiv / Year: 2023
Title: Cryo-EM structures of a synthetic antibody against 22 kDa claudin-4 reveal its complex with
Authors: Erramilli, S.K. / Dominik, P.K. / Ogbu, C.P. / Kossiakoff, A.A. / Vecchio, A.J.
History
DepositionSep 12, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 27, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 4, 2023Group: Database references / Category: pdbx_database_related / Item: _pdbx_database_related.details
Revision 1.2Dec 13, 2023Group: Derived calculations / Structure summary / Category: chem_comp / entity / pdbx_entity_nonpoly
Item: _chem_comp.name / _chem_comp.pdbx_synonyms ..._chem_comp.name / _chem_comp.pdbx_synonyms / _entity.pdbx_description / _pdbx_entity_nonpoly.name
Revision 1.3Jun 26, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.pdbx_database_id_DOI / _citation.title / _citation.year / _em_admin.last_update
Revision 1.4Jul 3, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.page_last / _citation.pdbx_database_id_PubMed ..._citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update
Revision 1.5Oct 30, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Claudin-4
B: Heat-labile enterotoxin B chain
L: COP-1 sFab Light Chain
H: COP-1 sFab Heavy Chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)88,9875
Polymers87,9824
Non-polymers1,0051
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 22613.852 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CLDN4, CPER, CPETR1, WBSCR8 / Plasmid: pFastBac1 / Cell line (production host): Tn5 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: O14493
#2: Protein Heat-labile enterotoxin B chain


Mass: 14044.594 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridium perfringens (bacteria) / Gene: cpe / Plasmid: pFastBac1 / Cell line (production host): Tn5 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P01558
#3: Antibody COP-1 sFab Light Chain


Mass: 23258.783 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#4: Antibody COP-1 sFab Heavy Chain


Mass: 28064.498 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#5: Chemical ChemComp-AV0 / Lauryl Maltose Neopentyl Glycol / 2,2-didecylpropane-1,3-bis-b-D-maltopyranoside / 2-decyl-2-{[(4-O-alpha-D-glucopyranosyl-beta-D-glucopyranosyl)oxy]methyl}dodecyl4-O-alpha-D-glucopyranosyl-beta-D-glucopyranoside


Mass: 1005.188 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C47H88O22
Has ligand of interestN
Has protein modificationY

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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 complex with Clostridium perfringens enterotoxin C-terminal domain and sFab COP-1
Type: COMPLEX
Details: Assembled complex of 4 proteins (Fab is 2 proteins) expressed from insect cells and E coli
Entity ID: #1-#4 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.1 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.4 / Details: 20 mM Hepes pH 7.4, 100 mM NaCl, and 0.003% LMNG
SpecimenConc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 92000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1073
EM imaging opticsEnergyfilter name: GIF Bioquantum

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2SerialEMimage acquisition
4cryoSPARCCTF correction
7PHENIXmodel fitting
9PHENIX1.20.1_4487:model refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 37586
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 5.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 37296 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 7kp4

7kp4


Accession code: 7kp4 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0126782
ELECTRON MICROSCOPYf_angle_d1.1929227
ELECTRON MICROSCOPYf_dihedral_angle_d4.928983
ELECTRON MICROSCOPYf_chiral_restr0.0641058
ELECTRON MICROSCOPYf_plane_restr0.0061155

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