To our surprise, the Stx1 L41Q mutant showed the weakest glycolipid binding of all B-subunits tested. This suggests that Stx1 B-subunits are capable of binding to glycolipids only as a stable pentamer. Stx2 B-subunits on the other hand can bind to glycolipids even in lower order oligomeric states. The Hill coefficients for Gb3 binding of the B-subunits were significantly different. Whereas Stx1 B-subunits bound to Gb3+ PC+Ch with a h value of 1 suggesting no cooperativity, Stx2a Bsubunits bound with a h value of 2.4 suggesting strong positive cooperativity. Previous studies by AUC showed that at high concentrations Stx2a B-subunits predominantly exist as pentamers, while a small proportion exists in the form of lower order oligomers. On the other hand, predominantly lower order oligomers exist at concentrations lower than 2 mM. Positive binding cooperativity observed with Stx2 B-subunits suggests that binding of these lower order oligomers may occur in two steps, initially B-subunits bind as monomers, and binding of one Bsubunit promotes binding of additional B-subunits to form higher order oligomers, ultimately forming pentamers. Since the pentamer formed by Stx1 B-subunits is more stable, this effect is not seen as prominently as with Stx2 B-subunits. Overall, this suggests that the B-subunits of Stx are capable of associating at the glycolipid interface. Holotoxins of Stx2 variants bound only to the intact glycolipid and no binding was observed to Lyso-Gb3, which lacked carbonyl and a fatty acid chain of Gb3. On the other hand, Stx1 holotoxin and Stx2a B-subunits, irrespective of the pentamer stabilities, did not differentiate between Gb3 and Lyso-Gb3, suggesting that the B-subunits are flexible about fatty acid requirement. Crystal structures of Stx holotoxins show that the C-terminus of A-subunit of Stx2 extends through the pore formed by the B-pentamer and could occlude receptor binding to a region defined as site 3 in Stx1. L-Arginine Consistently, in the recently reported co-crystal structure only two NAcPk disaccharide densities were reported on the Bsubunit of Stx2a holotoxin. It was speculated that the Asubunit interfered with binding to the glycan, which lacked the ceramide. It is therefore Iloprost possible that the ceramide portion of Gb3 is important for engaging the A-tail of Stx2a thereby opening glycan-binding sites on the B-subunits. Currently we are purifying Stx A-subunits to determine whether the A-subunits are capable of interacting with the glycolipids.