Unraveling the Molecular Basis of Cooperativity in Cholera Toxin–Glycan Interactions

). Stepwise apparent affinities for the primary sites reveal positive cooperativity that strengthens with both ligand occupancy and temperature. Van't Hoff analysis shows that this temperature-enhanced cooperativity is predominantly entropy driven. Mechanistic insight from temperature replica exchange molecular dynamics simulations shows that ligand binding at one primary site restrains a loop on an adjacent subunit (counterclockwise), widening its binding site. This prestructuring progressively lowers the unfavorable conformational entropy penalty of binding, independent of the order of subunit occupancy. In contrast, sequential ligand binding at the secondary sites exhibits negligible cooperativity except at the highest temperatures, although the enthalpic and entropic contributions are comparable in magnitude to those for primary-site binding, suggesting a shared energetic framework. Together, these results provide detailed thermodynamic and structural insight into cooperative GBP-glycan interactions and establish an integrated VT-nMS and molecular dynamics framework for quantitatively probing cooperative ligand binding in complex biomolecular systems.