+Open data
-Basic information
Entry | Database: PDB / ID: 6ar6 | ||||||||||||
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Title | Clostridioides difficile toxinB with DLD-4 darpin | ||||||||||||
Components |
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Keywords | TOXIN/PROTEIN BINDING / TOXIN-PROTEIN BINDING complex | ||||||||||||
Function / homology | Function and homology information glucosyltransferase activity / host cell cytosol / Transferases; Glycosyltransferases; Hexosyltransferases / cysteine-type peptidase activity / host cell endosome membrane / toxin activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / lipid binding / host cell plasma membrane / proteolysis ...glucosyltransferase activity / host cell cytosol / Transferases; Glycosyltransferases; Hexosyltransferases / cysteine-type peptidase activity / host cell endosome membrane / toxin activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / lipid binding / host cell plasma membrane / proteolysis / extracellular region / membrane / metal ion binding Similarity search - Function | ||||||||||||
Biological species | Clostridioides difficile (bacteria) Drosophila melanogaster (fruit fly) | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 9 Å | ||||||||||||
Authors | Zhang, J. / Jiang, M. | ||||||||||||
Funding support | United States, 3items
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Citation | Journal: PLoS Biol / Year: 2019 Title: Selection and characterization of ultrahigh potency designed ankyrin repeat protein inhibitors of C. difficile toxin B. Authors: Rudo Simeon / Mengqiu Jiang / Ana M Chamoun-Emanuelli / Hua Yu / Yongrong Zhang / Ran Meng / Zeyu Peng / Joanita Jakana / Junjie Zhang / Hanping Feng / Zhilei Chen / Abstract: Clostridium difficile infection (CDI) is a major nosocomial disease associated with significant morbidity and mortality. The pathology of CDI stems primarily from the 2 C. difficile-secreted ...Clostridium difficile infection (CDI) is a major nosocomial disease associated with significant morbidity and mortality. The pathology of CDI stems primarily from the 2 C. difficile-secreted exotoxins-toxin A (TcdA) and toxin B (TcdB)-that disrupt the tight junctions between epithelial cells leading to the loss of colonic epithelial barrier function. Here, we report the engineering of a series of monomeric and dimeric designed ankyrin repeat proteins (DARPins) for the neutralization of TcdB. The best dimeric DARPin, DLD-4, inhibited TcdB with a half maximal effective concentration (EC50) of 4 pM in vitro, representing an approximately 330-fold higher potency than the Food and Drug Administration (FDA)-approved anti-TcdB monoclonal antibody bezlotoxumab in the same assay. DLD-4 also protected mice from a toxin challenge in vivo. Cryo-electron microscopy (cryo-EM) studies revealed that the 2 constituent DARPins of DLD-4-1.4E and U3-bind the central and C-terminal regions of the delivery domain of TcdB. Competitive enzyme-linked immunosorbent assay (ELISA) studies showed that the DARPins 1.4E and U3 interfere with the interaction between TcdB and its receptors chondroitin sulfate proteoglycan 4 (CSPG4) and frizzled class receptor 2 (FZD2), respectively. Our cryo-EM studies revealed a new conformation of TcdB (both apo- and DARPin-bound at pH 7.4) in which the combined repetitive oligopeptides (CROPS) domain points away from the delivery domain. This conformation of the CROPS domain is in stark contrast to that seen in the negative-stain electron microscopy (EM) structure of TcdA and TcdB at the same pH, in which the CROPS domain bends toward and "kisses" the delivery domain. The ultrapotent anti-TcdB molecules from this study serve as candidate starting points for CDI drug development and provide new biological tools for studying the pathogenicity of C. difficile. The structural insights regarding both the "native" conformation of TcdB and the putative sites of TcdB interaction with the FZD2 receptor, in particular, should help accelerate the development of next-generation anti-C. difficile toxin therapeutics. | ||||||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6ar6.cif.gz | 94.3 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6ar6.ent.gz | 48.2 KB | Display | PDB format |
PDBx/mmJSON format | 6ar6.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ar/6ar6 ftp://data.pdbj.org/pub/pdb/validation_reports/ar/6ar6 | HTTPS FTP |
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-Related structure data
Related structure data | 8898MC 8897C M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 235481.438 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Clostridioides difficile (bacteria) / Gene: toxB, tcdB / Production host: Escherichia coli (E. coli) References: UniProt: P18177, Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases |
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#2: Protein | Mass: 34299.195 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Production host: Escherichia coli (E. coli) |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: ternary complex of tcdB and DLD-4 darpin / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT |
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Source (natural) | Organism: Clostridioides difficile (bacteria) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 7.4 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Tecnai F20 / Image courtesy: FEI Company | |||||||||||||||||||||
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EM imaging |
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Image recording |
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-Processing
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION |
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Symmetry | Point symmetry: C1 (asymmetric) |
3D reconstruction | Resolution: 9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 84420 / Symmetry type: POINT |