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- EMDB-45749: Cryo-EM structure of human claudin-4 complex with Clostridium per... -

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Basic information

Entry
Database: EMDB / ID: EMD-45749
TitleCryo-EM structure of human claudin-4 complex with Clostridium perfringens enterotoxin, sFab COP-1, and Nanobody
Map data
Sample
  • Complex: Human claudin-4 complex with Clostridium perfringens enterotoxin, sFab COP-1, and Nanobody
    • Protein or peptide: Claudin-4
    • Protein or peptide: Heat-labile enterotoxin B chain
    • Protein or peptide: COP-1 sFab Heavy Chain
    • Protein or peptide: Anti-Fab Nanobody
    • Protein or peptide: COP-1 sFab Light Chain
  • Ligand: Lauryl Maltose Neopentyl Glycol
  • Ligand: water
Keywordsclaudin / Fab / enterotoxin / nanobody / MEMBRANE PROTEIN
Function / homology
Function and homology information


paracellular transport / positive regulation of metallopeptidase activity / 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 / chloride channel activity ...paracellular transport / positive regulation of metallopeptidase activity / 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 / chloride channel activity / 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 / 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
Claudin-4 / Heat-labile enterotoxin B chain
Similarity search - Component
Biological speciessynthetic construct (others) / Homo sapiens (human) / Clostridium perfringens (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.83 Å
AuthorsVecchio AJ
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: Structure / Year: 2024
Title: Cryo-EM structures of Clostridium perfringens enterotoxin bound to its human receptor, claudin-4.
Authors: Sewwandi S Rathnayake / Satchal K Erramilli / Anthony A Kossiakoff / Alex J Vecchio /
Abstract: Clostridium perfringens enterotoxin (CpE) causes prevalent and deadly gastrointestinal disorders. CpE binds to receptors called claudins on the apical surfaces of small intestinal epithelium. ...Clostridium perfringens enterotoxin (CpE) causes prevalent and deadly gastrointestinal disorders. CpE binds to receptors called claudins on the apical surfaces of small intestinal epithelium. Claudins normally regulate paracellular transport but are hijacked from doing so by CpE and are instead led to form claudin/CpE complexes. Claudin/CpE complexes are the building blocks of oligomeric β-barrel pores that penetrate the plasma membrane and induce gut cytotoxicity. Here, we present the structures of CpE in complex with its native claudin receptor in humans, claudin-4, using cryogenic electron microscopy. The structures reveal the architecture of the claudin/CpE complex, the residues used in binding, the orientation of CpE relative to the membrane, and CpE-induced changes to claudin-4. Further, structures and modeling allude to the biophysical procession from claudin/CpE complexes to cytotoxic β-barrel pores during pathogenesis. In full, this work proposes a model of claudin/CpE assembly and provides strategies to obstruct its formation to treat CpE diseases.
History
DepositionJul 15, 2024-
Header (metadata) releaseAug 7, 2024-
Map releaseAug 7, 2024-
UpdateOct 30, 2024-
Current statusOct 30, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45749.png

Downloads & links

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Map

FileDownload / File: emd_45749.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 360 pix.
= 297.72 Å		0.83 Å/pix.
x 360 pix.
= 297.72 Å		0.83 Å/pix.
x 360 pix.
= 297.72 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.827 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.26698318 - 0.582481
Average (Standard dev.)-0.0003027218 (±0.009423368)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 297.72 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_45749_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_45749_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 complex with Clostridium perfringens enterotoxin,...

EntireName: Human claudin-4 complex with Clostridium perfringens enterotoxin, sFab COP-1, and Nanobody
Components
  • Complex: Human claudin-4 complex with Clostridium perfringens enterotoxin, sFab COP-1, and Nanobody
    • Protein or peptide: Claudin-4
    • Protein or peptide: Heat-labile enterotoxin B chain
    • Protein or peptide: COP-1 sFab Heavy Chain
    • Protein or peptide: Anti-Fab Nanobody
    • Protein or peptide: COP-1 sFab Light Chain
  • Ligand: Lauryl Maltose Neopentyl Glycol
  • Ligand: water

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Supramolecule #1: Human claudin-4 complex with Clostridium perfringens enterotoxin,...

SupramoleculeName: Human claudin-4 complex with Clostridium perfringens enterotoxin, sFab COP-1, and Nanobody
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5 / Details: 5-protein complex
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 120 KDa

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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: 22.234332 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: GSMASMGLQV MGIALAVLGW LAVMLCCALP MWRVTAFIGS NIVTSQTIWE GLWMNCVVQS TGQMQCKVYD SLLALPQDLQ AARALVIIS IIVAALGVLL SVVGGKCTNC LEDESAKAKT MIVAGVVFLL AGLMVIVPVS WTAHNIIQDF YNPLVASGQK R EMGASLYV ...String:
GSMASMGLQV MGIALAVLGW LAVMLCCALP MWRVTAFIGS NIVTSQTIWE GLWMNCVVQS TGQMQCKVYD SLLALPQDLQ AARALVIIS IIVAALGVLL SVVGGKCTNC LEDESAKAKT MIVAGVVFLL AGLMVIVPVS WTAHNIIQDF YNPLVASGQK R EMGASLYV GWAASGLLLL GGGLLCCNCP PRTDKPYSAK YSAARSAAAS NYV

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 / Details: Trypsin treated CpE / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Clostridium perfringens (bacteria)
Molecular weightTheoretical: 33.000582 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: TPINITNSNS NLSDGLYVID KGDGWILGEP SVVSSQILNP NETGTFSQSL TKSKEVSINV NFSVGFTSEF IQASVEYGFG ITIGEQNTI ERSVSTTAGP NEYVYYKVYA TYRKYQAIRI SHGNISDDGS IYKLTGIWLS KTSADSLGNI DQGSLIETGE R CVLTVPST ...String:
TPINITNSNS NLSDGLYVID KGDGWILGEP SVVSSQILNP NETGTFSQSL TKSKEVSINV NFSVGFTSEF IQASVEYGFG ITIGEQNTI ERSVSTTAGP NEYVYYKVYA TYRKYQAIRI SHGNISDDGS IYKLTGIWLS KTSADSLGNI DQGSLIETGE R CVLTVPST DIEKEILDLA AATERLNLTD ALNSNPAGNL YDWRSSNSYP WTQKLNLHLT ITATGQKYRI LASKIVDFNI YS NNFNNLV KLEQSLGDGV KDHYVDISLD AGQYVLVMKA NSSYSGNYPY SILFQKFGLV PR

UniProtKB: Heat-labile enterotoxin B chain

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Macromolecule #3: COP-1 sFab Heavy Chain

MacromoleculeName: COP-1 sFab Heavy Chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 28.064498 KDa
SequenceString: MKKNIAFLLA SMFVFSIATN AYAEISEVQL VESGGGLVQP GGSLRLSCAA SGFNFSSSYI HWVRQAPGKG LEWVASISSS SGSTSYADS VKGRFTISAD TSKNTAYLQM NSLRAEDTAV YYCARWFHPW WWWEYLFRGA IDYWGQGTLV TVSSASTKGP S VFPLAPSS ...String:
MKKNIAFLLA SMFVFSIATN AYAEISEVQL VESGGGLVQP GGSLRLSCAA SGFNFSSSYI HWVRQAPGKG LEWVASISSS SGSTSYADS VKGRFTISAD TSKNTAYLQM NSLRAEDTAV YYCARWFHPW WWWEYLFRGA IDYWGQGTLV TVSSASTKGP S VFPLAPSS KSTSGGTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSVVTVPSSS LGTQTYICNV NH KPSNTKV DKKVEPKSCD KTHT

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Macromolecule #4: Anti-Fab Nanobody

MacromoleculeName: Anti-Fab Nanobody / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 13.175438 KDa
SequenceString:
GSVQLQESGG GLVQPGGSLR LSCAASGRTI SRYAMSWFRQ APGKEREFVA VARRSGDGAF YADSVQGRFT VSRDDAKNTV YLQMNSLKP EDTAVYYCAI DSDTFYSGSY DYWGQGTQVT VS

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Macromolecule #5: COP-1 sFab Light Chain

MacromoleculeName: COP-1 sFab Light Chain / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.794859 KDa
SequenceString: MKKNIAFLLA SMFVFSIATN AYASDIQMTQ SPSSLSASVG DRVTITCRAS QSVSSAVAWY QQKPGKAPKL LIYSASSLYS GVPSRFSGS RSGTDFTLTI SSLQPEDFAT YYCQQSSSSL ITFGQGTKVE IKRTVAAPSV FIFPPSDSQL KSGTASVVCL L NNFYPREA ...String:
MKKNIAFLLA SMFVFSIATN AYASDIQMTQ SPSSLSASVG DRVTITCRAS QSVSSAVAWY QQKPGKAPKL LIYSASSLYS GVPSRFSGS RSGTDFTLTI SSLQPEDFAT YYCQQSSSSL ITFGQGTKVE IKRTVAAPSV FIFPPSDSQL KSGTASVVCL L NNFYPREA KVQWKVDNAL QSGNSQESVT EQDSKDSTYS LSSTLTLSKA DYEKHKVYAC EVTHQGLSSP VTKSFNRGEC

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Macromolecule #6: Lauryl Maltose Neopentyl Glycol

MacromoleculeName: Lauryl Maltose Neopentyl Glycol / type: ligand / ID: 6 / Number of copies: 1 / Formula: AV0
Molecular weightTheoretical: 1.005188 KDa
Chemical component information

ChemComp-AV0:
Lauryl Maltose Neopentyl Glycol

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Macromolecule #7: water

MacromoleculeName: water / type: ligand / ID: 7 / Number of copies: 2 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
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

Concentration5.6 mg/mL
BufferpH: 7.4
Component:
ConcentrationNameFormula
20.0 mMHepes pH 7.4
100.0 mMNaClNaCl
0.003 %LMNG

Details: 20 mM Hepes pH 7.4, 100 mM NaCl, and 0.003% LMNG
GridModel: UltrAuFoil / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
Details: glow-discharged for 30 seconds at 20 W using a Solarus 950 (Gatan) plasma cleaner
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum
SoftwareName: EPU
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 6774 / Average electron dose: 70.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.1 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 5766325
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

8u4v

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.83 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4) / Number images used: 431680
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 4.4)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

84uv

source_name: PDB, initial_model_type: experimental model

8u5f

source_name: PDB, initial_model_type: experimental model
SoftwareName: UCSF Chimera
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9cmi:
Cryo-EM structure of human claudin-4 complex with Clostridium perfringens enterotoxin, sFab COP-1, and Nanobody

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