Cooperative ligand binding is an important determinant of specificity and regulation in biomolecular complexes. Yet, its prevalence and mechanistic basis in glycan-binding proteins (GBPs) remain unclear. Here, we present the first quantitative analysis of the temperature dependence of cooperative glycan binding. Variable-temperature native mass spectrometry (VT-nMS) resolved sequential ligand binding to either the five primary or the five secondary sites of the cholera toxin B subunit homopentamer (CTB5). 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.

ALT: Alexis Rose: I Don't Know You Tell Me (Schitt's Creek)

Led by Duong Bui, Assistant Professor in Biochemistry, Departments of Chemistry and Biology

Slow mixing mode native mass spectrometry (SLOMO-nMS), which monitors the mixing of layered solutions within a nanoflow electrospray ionization (nanoESI) emitter, enables the accurate quantification o...

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Slow mixing mode native mass spectrometry (SLOMO-nMS), which monitors the mixing of layered solutions within a nanoflow electrospray ionization (nanoESI) emitter, enables robust quantification of biom...

Ambient and direct mass spectrometry (MS) methods are becoming increasingly used for the rapid analysis of food, beverage and agricultural samples. Novel ionization approaches combined with targeted,...

Interactions between glycan-binding proteins (GBPs) and carbohydrates (glycans) are essential to many biological processes relevant to human health and disease. For most GBPs, however, their glycan i...

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