Combined repetitive oligopeptides of clostridium difficile toxin A counteract premature cleavage of the glucosyl-transferase domain by...

 

Alexandra Olling, Corinna Hüls, Sebastian Goy, Mirco Müller, Simon Krooss, Isa Rudolf, Helma Tatge and Ralf Gerhard

Toxins
2014 vol: 6 issue: 7 pp: 2162-2176 doi: 10.3390/toxins6072162

Abstract

Toxin A (TcdA) and B (TcdB) from Clostridium difficile enter host cells by receptor-mediated endocytosis. A prerequisite for proper toxin action is the intracellular release of the glucosyltransferase domain by an inherent cysteine protease, which is allosterically activated by inositol hexaphosphate (IP6). We found that in in vitro assays, the C-terminally-truncated TcdA1-1065 was more efficient at IP6-induced cleavage compared with full-length TcdA. We hypothesized that the C-terminally-located combined repetitive oligopeptides (CROPs) interact with the N-terminal part of the toxin, thereby preventing autoproteolysis. Glutathione-S-transferase (GST) pull-down assays and microscale thermophoresis confirmed binding between the CROPs and the glucosyltransferase (TcdA1-542) or intermediate (TcdA1102-1847) domain of TcdA, respectively. This interaction between the N- and C-terminus was not found for TcdB. Functional assays revealed that TcdB was more susceptible to inactivation by extracellular IP6-induced cleavage. In vitro autoprocessing and inactivation of TcdA, however, significantly increased, either by acidification of the surrounding milieu or following exchange of its CROP domain by the homologous CROP domain of TcdB. Thus, TcdA CROPs contribute to the stabilization and protection of toxin conformation in addition to function as the main receptor binding domain.

View Publication

Topics: Clostridium difficile toxin, Domain interaction, Autoprocessing, Cytotoxicity, Monolith – MicroScale Thermophoresis, MST, Proteins, Publications

 

 

Previous Article
Protein HESylation for half-life extension: synthesis, characterization and pharmacokinetics of HESylated anakinra
Protein HESylation for half-life extension: synthesis, characterization and pharmacokinetics of HESylated anakinra

Up next
Structural basis of malaria parasite lysyl-tRNA synthetase inhibition by cladosporin
Structural basis of malaria parasite lysyl-tRNA synthetase inhibition by cladosporin

Ready to characterize your most challenging interactions?

Discover tools to measure binding affinity

Learn more