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Title: Structure of the cell-binding component of the Clostridium difficile binary toxin reveals a di-heptamer macromolecular assembly

Abstract

TargetingClostridium difficileinfection is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulentC. difficilestrains often have a binary toxin termed theC. difficiletoxin, in addition to the enterotoxins TsdA and TsdB. TheC. difficiletoxin has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of single-particle cryoelectron microscopy, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, 2 di-heptamer structures for activated CDTb (1.0 MDa) were solved at atomic resolution, including a symmetric (SymCDTb; 3.14 Å) and an asymmetric form (AsymCDTb; 2.84 Å). Roles played by 2 receptor-binding domains of activated CDTb were of particular interest since the receptor-binding domain 1 lacks sequence homology to any other known toxin, and the receptor-binding domain 2 is completely absent in other well-studied heptameric toxins (i.e., anthrax). ForAsymCDTb, a Ca2+binding site was discovered in the first receptor-binding domain that is important for its stability, and the second receptor-binding domain was found to be critical for host cell toxicity and the di-heptamer fold for both forms of activated CDTb. Together, these studies represent a starting point for developing structure-based drug-design strategies to targetmore » the most severe strains ofC. difficile.« less

Authors:
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Publication Date:
Research Org.:
SLAC National Accelerator Lab., Menlo Park, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES)
OSTI Identifier:
1581008
Alternate Identifier(s):
OSTI ID: 1625064
Grant/Contract Number:  
AC02-76SF00515
Resource Type:
Published Article
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Volume: 117 Journal Issue: 2; Journal ID: ISSN 0027-8424
Publisher:
Proceedings of the National Academy of Sciences
Country of Publication:
United States
Language:
English
Subject:
Science & Technology - Other Topics

Citation Formats

Xu, Xingjian, Godoy-Ruiz, Raquel, Adipietro, Kaylin A., Peralta, Christopher, Ben-Hail, Danya, Varney, Kristen M., Cook, Mary E., Roth, Braden M., Wilder, Paul T., Cleveland, Thomas, Grishaev, Alexander, Neu, Heather M., Michel, Sarah L. J., Yu, Wenbo, Beckett, Dorothy, Rustandi, Richard R., Lancaster, Catherine, Loughney, John W., Kristopeit, Adam, Christanti, Sianny, Olson, Jessica W., MacKerell, Alexander D., Georges, Amedee des, Pozharski, Edwin, and Weber, David J. Structure of the cell-binding component of the Clostridium difficile binary toxin reveals a di-heptamer macromolecular assembly. United States: N. p., 2020. Web. doi:10.1073/pnas.1919490117.
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Xu, Xingjian, Godoy-Ruiz, Raquel, Adipietro, Kaylin A., Peralta, Christopher, Ben-Hail, Danya, Varney, Kristen M., Cook, Mary E., Roth, Braden M., Wilder, Paul T., Cleveland, Thomas, Grishaev, Alexander, Neu, Heather M., Michel, Sarah L. J., Yu, Wenbo, Beckett, Dorothy, Rustandi, Richard R., Lancaster, Catherine, Loughney, John W., Kristopeit, Adam, Christanti, Sianny, Olson, Jessica W., MacKerell, Alexander D., Georges, Amedee des, Pozharski, Edwin, & Weber, David J. Structure of the cell-binding component of the Clostridium difficile binary toxin reveals a di-heptamer macromolecular assembly. United States. doi:10.1073/pnas.1919490117.
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Xu, Xingjian, Godoy-Ruiz, Raquel, Adipietro, Kaylin A., Peralta, Christopher, Ben-Hail, Danya, Varney, Kristen M., Cook, Mary E., Roth, Braden M., Wilder, Paul T., Cleveland, Thomas, Grishaev, Alexander, Neu, Heather M., Michel, Sarah L. J., Yu, Wenbo, Beckett, Dorothy, Rustandi, Richard R., Lancaster, Catherine, Loughney, John W., Kristopeit, Adam, Christanti, Sianny, Olson, Jessica W., MacKerell, Alexander D., Georges, Amedee des, Pozharski, Edwin, and Weber, David J. Thu . "Structure of the cell-binding component of the Clostridium difficile binary toxin reveals a di-heptamer macromolecular assembly". United States. doi:10.1073/pnas.1919490117.
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@article{osti_1581008,
title = {Structure of the cell-binding component of the Clostridium difficile binary toxin reveals a di-heptamer macromolecular assembly},
author = {Xu, Xingjian and Godoy-Ruiz, Raquel and Adipietro, Kaylin A. and Peralta, Christopher and Ben-Hail, Danya and Varney, Kristen M. and Cook, Mary E. and Roth, Braden M. and Wilder, Paul T. and Cleveland, Thomas and Grishaev, Alexander and Neu, Heather M. and Michel, Sarah L. J. and Yu, Wenbo and Beckett, Dorothy and Rustandi, Richard R. and Lancaster, Catherine and Loughney, John W. and Kristopeit, Adam and Christanti, Sianny and Olson, Jessica W. and MacKerell, Alexander D. and Georges, Amedee des and Pozharski, Edwin and Weber, David J.},
abstractNote = {TargetingClostridium difficileinfection is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulentC. difficilestrains often have a binary toxin termed theC. difficiletoxin, in addition to the enterotoxins TsdA and TsdB. TheC. difficiletoxin has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of single-particle cryoelectron microscopy, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, 2 di-heptamer structures for activated CDTb (1.0 MDa) were solved at atomic resolution, including a symmetric (SymCDTb; 3.14 Å) and an asymmetric form (AsymCDTb; 2.84 Å). Roles played by 2 receptor-binding domains of activated CDTb were of particular interest since the receptor-binding domain 1 lacks sequence homology to any other known toxin, and the receptor-binding domain 2 is completely absent in other well-studied heptameric toxins (i.e., anthrax). ForAsymCDTb, a Ca2+binding site was discovered in the first receptor-binding domain that is important for its stability, and the second receptor-binding domain was found to be critical for host cell toxicity and the di-heptamer fold for both forms of activated CDTb. Together, these studies represent a starting point for developing structure-based drug-design strategies to target the most severe strains ofC. difficile.},
doi = {10.1073/pnas.1919490117},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 2,
volume = 117,
place = {United States},
year = {2020},
month = {1}
}
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DOI: 10.1073/pnas.1919490117
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Figures / Tables:

Fig. 1 Fig. 1: Functional studies of the RBD2 of CDTb. (A) Cellular toxicity upon the addition of CDTa to Vero cells in the presence of active CDTb (●) or active CDTb lacking the second RBD (i.e., CDTbΔRBD2; ■). The TC50 of CDTa is 150 ± 40 pM (n = 8 independentmore » experiments, ± SD) when active CDTb is present. Little or no toxicity is observed when CDTa is added Vero cells with active CDTbΔRBD2 even at the highest concentrations (≥10 nM; n = 3). For simplicity, the x axis presented using the CDTa concentration, but each experiment contains a 7x concentration of active CDTb or CDTbΔRBD2, as previously described. (B) A representative dominant-negative experiment showing the effect of adding the isolated RBD2 of CDTb (residues 757 to 876) into a Vero cell toxicity assay with 500 pM binary toxin. These data illustrate that the isolated RBD2 inhibits cellular toxicity, as a dominant-negative (IC50 = 20 ± 10 nM; n = 3) calculated using a 4-parameter logistic regression analysis. All data are plotted versus the normalized fluorescence of Alexa Fluor 488 Phalloidin, which selectively labels F-actin filaments; an increase in toxicity causes depolymerization of actin, causing a decrease in fluorescent signal.« less
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