Thermodynamics of Multivalent Carbohydrate−Lectin Cross-Linking Interactions: Importance of Entropy in the Bind and Jump Mechanism

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Abstract
The high affinity (Kd = 0.2 nM) of the soybean agglutinin (SBA), a tetrameric GalNAc specific lectin, for a modified form of porcine submaxillary mucin, a linear glycoprotein, with a molecular mass of ∼106 Da and ∼2300 GalNAcα1-O-Ser/Thr residues (Tn-PSM) has been ascribed to an internal diffusion mechanism that involves binding and jumping of the lectin from GalNAc to GalNAc residue of the mucin [Dam, T. K., et al. (2007) J. Biol. Chem. 282, 28256−28263]. Hill plot analysis of the raw ITC data shows increasing negative cooperativity, which correlates with an increasing number of lectin−mucin cross-linking interactions and decreasing favorable binding entropies. However, the affinity of bound SBA for other Tn-PSM molecules during cross-linking is much higher than that of free SBA for GalNAcα1-O-Ser, a monovalent analogue. The high affinity of bound SBA for GalNAc residues on other Tn-PSM molecules appears to be due to the favorable entropy of binding associated with the internal diffusion mechanism. Furthermore, the increasing negative cooperativity of SBA binding to Tn-PSM correlates with a decreasing level of internal diffusion of the lectin on the mucin as cross-linking occurs. These findings indicate the importance of the internal diffusion mechanism in generating large, favorable entropies of binding that drive lectin−mucin cross-linking interactions. The results are important for understanding the energetics of lectin−mucin cross-linking interactions that are associated with biological signaling on the surface of cells and the role of the internal diffusion mechanism in ligand-biopolymer interactions in general.