The proposed project uses crystallographic studies to unravel the interactions of two bacterial toxins, cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT), with a range of natural substrates and interacting proteins, as well as with designed ligands and inhibitors. The severe infectious diarrhea caused by CT may result in death within a few hours, while the milder diarrhea caused by enterotoxigenic LT-producing E. coli is mainly a traveler's discomfort for inhabitants of industrialized countries, but a major cause of infant death in the third world. CT and LT are remarkably sophisticated hetero-hexameric AB5 toxins that have many intriguing yet unresolved properties concerning their entry into the cytoplasm of the host cell, their catalytic mechanism, and the mode of secretion of CT from Vibrio cholerae. Based on significant results in the previous grant period we propose in the coming period to tackle, in collaboration with several groups with complementary expertise, structural studies to elucidate the following regarding CT and LT: 1. the mode of binding the substrates NAD and arginine-containing peptides, and the enzymatic mechanism of the ADP-ribosylating A-subunit, based on our recent success in determining the three-dimensional structure of a constitutively active form of CT. This will include a just initiated structure-based drug design collaborative project employing novel guanidine-based chemistry aimed at blocking the A-subunit; 2. the interactions of CT and LT with host proteins such as ADP-Ribosylation Factors (ARFs); 3. the design of ultra-high affinity GM1 receptor antagonists of the B-pentamer by employing multi-ring system-containing monovalent ligands and new "linkers" to obtain a next generation of our successful modular pentavalent and decavalent macroligands; 4. the structure of EpsC, a periplasmic inner membrane-anchored protein from the extracellular protein secretion apparatus in V. cholerae, and its interactions with CT; 5. the structure of the multimeric pore protein EpsD in the outer membrane of V. cholerae and its interactions with EpsC, and possibly, with cholera toxin.