Crystal structures reveal that Lewis-x and fucose bind to secondary cholera toxin binding site – in contrast to fucosyl-GM1

Cholera is a life-threatening diarrhoeal disease caused by the human pathogen Vibrio cholerae . Infection occurs after ingestion of the bacteria, which colonize the human small intestine and secrete their major virulence factor - the cholera toxin (CT). Recent studies suggest that the GM1 receptor may not be the only target of the CT, and that fucosylated receptors such as Lewisx (Lex) and histo-blood group antigens may also be important for cellular uptake and toxicity. However, where and how Lex binds to the CT remains unclear. Here we report the high-resolution crystal structure (1.5 Å) of the receptor-binding B-subunit of the CT bound to the Lex trisaccharide, and present matching SPR data for CT holotoxins. Lex, and also L-fucose alone (at 500-fold molar excess), bind to the secondary binding site of the toxin, distinct from the GM1 binding site. In contrast, fucosyl-GM1 mainly binds to the primary binding site due to high-affinity interactions of its GM1 core. The two binding sites are likely connected by allosteric cross-talk, potentially affecting toxin uptake. We also discuss why secretors are protected from severe cholera. Author summary Cholera is a severe diarrhoeal disease that is still a major killer in many parts of the world, especially in regions struck by natural disasters and wars. However, some individuals experience milder cholera symptoms. These so-called ‘secretors’, who have blood group antigens also in their bodily fluids like their saliva and the slimy mucus layer covering their stomach and intestines, appear to be somewhat protected. Here we present detailed atomic structures of cholera toxin and quantitative binding data that give clues of the protective mechanisms. Interactions of the protein toxin with sugar molecules are of crucial importance both for toxicity and protection. In addition, we identify a new tool for biochemical studies, and lay the groundwork for the design of cholera drugs and vaccines that may save countless human lives.