4NP4

Clostridium difficile toxin B CROP domain in complex with FAB domains of neutralizing antibody bezlotoxumab


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.89 Å
  • R-Value Free: 0.232 
  • R-Value Work: 0.198 
  • R-Value Observed: 0.199 

wwPDB Validation   3D Report Full Report


This is version 1.4 of the entry. See complete history


Literature

Mechanism of Action and Epitopes of Clostridium difficile Toxin B-neutralizing Antibody Bezlotoxumab Revealed by X-ray Crystallography.

Orth, P.Xiao, L.Hernandez, L.D.Reichert, P.Sheth, P.R.Beaumont, M.Yang, X.Murgolo, N.Ermakov, G.DiNunzio, E.Racine, F.Karczewski, J.Secore, S.Ingram, R.N.Mayhood, T.Strickland, C.Therien, A.G.

(2014) J Biol Chem 289: 18008-18021

  • DOI: https://doi.org/10.1074/jbc.M114.560748
  • Primary Citation of Related Structures:  
    4NP4

  • PubMed Abstract: 

    The symptoms of Clostridium difficile infections are caused by two exotoxins, TcdA and TcdB, which target host colonocytes by binding to unknown cell surface receptors, at least in part via their combined repetitive oligopeptide (CROP) domains. A combination of the anti-TcdA antibody actoxumab and the anti-TcdB antibody bezlotoxumab is currently under development for the prevention of recurrent C. difficile infections. We demonstrate here through various biophysical approaches that bezlotoxumab binds to specific regions within the N-terminal half of the TcdB CROP domain. Based on this information, we solved the x-ray structure of the N-terminal half of the TcdB CROP domain bound to Fab fragments of bezlotoxumab. The structure reveals that the TcdB CROP domain adopts a β-solenoid fold consisting of long and short repeats and that bezlotoxumab binds to two homologous sites within the CROP domain, partially occluding two of the four putative carbohydrate binding pockets located in TcdB. We also show that bezlotoxumab neutralizes TcdB by blocking binding of TcdB to mammalian cells. Overall, our data are consistent with a model wherein a single molecule of bezlotoxumab neutralizes TcdB by binding via its two Fab regions to two epitopes within the N-terminal half of the TcdB CROP domain, partially blocking the carbohydrate binding pockets of the toxin and preventing toxin binding to host cells.


  • Organizational Affiliation

    From Merck & Co., Inc., Kenilworth, New Jersey 07023.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Toxin B275Clostridioides difficileMutation(s): 0 
UniProt
Find proteins for P18177 (Clostridioides difficile)
Explore P18177 
Go to UniProtKB:  P18177
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP18177
Sequence Annotations
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  • Reference Sequence
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Entity ID: 2
MoleculeChains Sequence LengthOrganismDetailsImage
bezlotoxumab heavy chainB [auth H],
C [auth I]
222Homo sapiensMutation(s): 0 
Entity Groups  
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Sequence Annotations
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  • Reference Sequence
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Entity ID: 3
MoleculeChains Sequence LengthOrganismDetailsImage
bezlotoxumab light chainD [auth L],
E [auth M]
215Homo sapiensMutation(s): 0 
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.89 Å
  • R-Value Free: 0.232 
  • R-Value Work: 0.198 
  • R-Value Observed: 0.199 
  • Space Group: P 1 21 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 79.413α = 90
b = 134.659β = 112.56
c = 102.579γ = 90
Software Package:
Software NamePurpose
JDirectordata collection
AMoREphasing
BUSTERrefinement
XDSdata reduction
SCALAdata scaling

Structure Validation

View Full Validation Report



Entry History 

Revision History  (Full details and data files)

  • Version 1.0: 2014-05-21
    Type: Initial release
  • Version 1.1: 2014-06-25
    Changes: Database references
  • Version 1.2: 2014-07-16
    Changes: Database references
  • Version 1.3: 2017-11-22
    Changes: Refinement description
  • Version 1.4: 2023-09-20
    Changes: Data collection, Database references, Refinement description