Hetero-multivalent binding of cholera toxin subunit B with glycolipid mixtures.

GM1 has generally been considered as the major receptor that binds to cholera toxin subunit B (CTB) due to its low dissociation constant. However, using a unique nanocube sensor technology, we have shown that CTB can also bind to other glycolipid receptors, fucosyl-GM1 and GD1b. Additionally, we have demonstrated that GM2 can contribute to CTB binding if present in a glycolipid mixture with a strongly binding receptor (GM1/fucosyl-GM1/GD1b). This hetero-multivalent binding result was unintuitive because the interaction between CTB and pure GM2 is negligible. We hypothesized that the reduced dimensionality of CTB-GM2 binding events is a major cause of the observed CTB binding enhancement. Once CTB has attached to a strong receptor, subsequent binding events are confined to a 2D membrane surface. Therefore, even a weak GM2 receptor could now participate in second or higher binding events because its surface reaction rate can be up to 104 times higher than the bulk reaction rate. To test this hypothesis, we altered the surface reaction rate by modulating the fluidity and heterogeneity of the model membrane. Decreasing membrane fluidity reduced the binding cooperativity between GM2 and a strong receptor. Our findings indicated a new protein-receptor binding assay, that can mimic complex cell membrane environment more accurately, is required to explore the inherent hetero-multivalency of the cell membrane. We have thus developed a new membrane perturbation protocol to efficiently screen receptor candidates involved in hetero-multivalent protein binding.