The TcdA and TcdB toxins from Clostridium difficile are amongst the largest toxins produced by bacterial pathogens (308 kDa and 270 kDa respectively). These toxins are single polypeptides, but their enzymatic, translocation and receptor binding activities are contained within domains that can be cloned and expressed separately. No structural information is available for any LCT toxin, which severely impedes our understanding of the mechanism of action of this toxin family. However, our preliminary data includes demonstration that the N-terminal glycosyltransferase and the C-terminal receptor binding domains can be purified from recombinant clones. We have also obtained crystals of a domain containing 14 of the 38 repeats within the C-terminal domain of TcdA, and we have an NMR solution structure for 5 of these repeat sequences. We can also visualise individual holotoxin molecules by cryo-electron microscopy.<P> In this application our aims are to obtain structures of domains of the TcdA and TcdB toxins:<OL> <LI> To express the N-terminal glycosyltransferase domains and the C-terminal receptor binding domains of TcdA and/or TcdB in E. coli and L. lactis and to prepare crystals and to determine their structures. <LI> To probe the binding of the toxins to putative carbohydrate ligands using ELISA assays and in solution using NMR. Co-crystallisation of receptor binding domains will be attempted with any putative carbohydrate ligand showing high affinity binding. <LI> To prepare native holotoxins from C. difficile and to obtain 10-15 Angstrom low resolution molecular envelopes of single molecules using cryo-electron microscopy, and to attempt to dock X-ray crystal structures to these molecular envelopes.</ol> The structural information obtained in the work will significantly increase our understanding of the mechanism of actions of this toxin family. It will lay the foundation for further work to explore the mechanisms of glycosylation of the GTP-ase substrates, the binding to host cell receptors and the process of translocation of the toxins from the host cell surface to the cytosol. It will also aid in exploiting these toxins as targets for therapy and for the design of novel vaccines.