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
Citation
Journal: Protein Sci / Year: 2025 Title: Structures of a synthetic antibody selected against and bound to the C-terminal domain of Clostridium perfringens enterotoxin. Authors: Chinemerem P Ogbu / Nicolas Manuel Goldbach / Martin Pacesa / Srajan Kapoor / Bruno E Correia / Alex J Vecchio / Abstract: Clostridium perfringens enterotoxin (CpE) causes cytotoxic gastrointestinal disease in mammalian epithelium by binding membrane protein receptors called claudins. Claudins direct the formation of ...Clostridium perfringens enterotoxin (CpE) causes cytotoxic gastrointestinal disease in mammalian epithelium by binding membrane protein receptors called claudins. Claudins direct the formation of cell/cell tight junctions through oligomerization and govern the transport of molecules between individual cells. CpE binds claudins through its C-terminal domain (cCpE) and induces cytotoxicity through its N-terminal domain. The non-toxic cCpE is a useful tool to study claudins, tight junctions, and for translational applications, such as increasing the permeability of restrictive tissues like the blood-brain barrier or selective targeting of claudin overexpressing cancers. Conversely, there are no specialized molecular tools to study CpE or cCpE, or to modulate or inhibit their functions. We previously reported the development of synthetic antigen-binding fragments (sFabs) that bind cCpE, and low-resolution structures of them bound to claudin/cCpE complexes. Here, we determine high-resolution structures of sFab COP-2 bound to cCpE using X-ray crystallography and cryogenic electron microscopy. The structures and biophysical findings provide the mechanism of COP-2 binding to cCpE and the molecular determinants driving their interactions. These insights can advance the design of new antibody-based tools from our COP-2 scaffold to study or alter cCpE function and give rise to a "Trojan horse" strategy that exploits cCpE's tight junction barrier disrupting function to selectively deliver conjugated therapeutics through normally impermeable tissues.
Evidence: gel filtration, The orientation in the crystal in crystal induced and not present in solution, only a single COP-2/cCpE is in solution as verified by SEC and cryo-EM
Mass: 26053.135 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#2: Antibody
COP-2Heavychain
Mass: 27799.080 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#3: Protein
Heat-labileenterotoxinBchain
Mass: 15114.945 Da / Num. of mol.: 3 / 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
Mass: 18.015 Da / Num. of mol.: 323 / Source method: isolated from a natural source / Formula: H2O
Has protein modification
Y
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Experimental details
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Experiment
Experiment
Method: X-RAY DIFFRACTION / Number of used crystals: 1
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Sample preparation
Crystal
Density Matthews: 2.91 Å3/Da / Density % sol: 63 %
Crystal grow
Temperature: 277.15 K / Method: vapor diffusion, sitting drop / pH: 6.6 Details: 200 mM sodium chloride, 1.75 M ammonium sulfate, and 100 mM sodium cacodylate pH 6.6
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Data collection
Diffraction
Mean temperature: 100 K / Serial crystal experiment: N