Binding Cooperativity Matters: A GM.sub.1-Like Ganglioside-Cholera Toxin B Subunit Binding Study Using a Nanocube-Based Lipid Bilayer Array

Protein-glycan recognition is often mediated by multivalent binding. These multivalent bindings can be further complicated by cooperative interactions between glycans and individual glycan binding subunits. Here we have demonstrated a nanocube-based lipid bilayer array capable of quantitatively elucidating binding dissociation constants, maximum binding capacity, and binding cooperativity in a high-throughput format. Taking cholera toxin B subunit (CTB) as a model cooperativity system, we studied both GM.sub.1 and GM.sub.1 -like gangliosides binding to CTB. We confirmed the previously observed CTB-GM.sub.1 positive cooperativity. Surprisingly, we demonstrated fucosyl-GM.sub.1 has approximately 7 times higher CTB binding capacity than GM.sub.1 . In order to explain this phenomenon, we hypothesized that the reduced binding cooperativity of fucosyl-GM.sub.1 caused the increased binding capacity. This was unintuitive, as GM.sub.1 exhibited higher binding avidity (16 times lower dissociation constant). We confirmed the hypothesis using a theoretical stepwise binding model of CTB. Moreover, by taking a mixture of fucosyl-GM.sub.1 and GM.sub.2, we observed the mild binding avidity fucosyl-GM.sub.1 activated GM.sub.2 receptors enhancing the binding capacity of the lipid bilayer surface. This was unexpected as GM.sub.2 receptors have negligible binding avidity in pure GM.sub.2 bilayers. These unexpected discoveries demonstrate the importance of binding cooperativity in multivalent binding mechanisms. Thus, quantitative analysis of multivalent protein-glycan interactions in heterogeneous glycan systems is of critical importance. Our user-friendly, robust, and high-throughput nanocube-based lipid bilayer array offers an attractive method for dissecting these complex mechanisms.