One of the reasons I love science is because sometimes jigsaw puzzles such as the cholera toxin B sub pentamer (CTB5) land on your desk... And here it is, fresh off the press ⤵️😎 Led by John Klassen and team at the wheel with the variable temp nMS 🍳 REMD analysis reviewed ⤵️ doi.org/10.1021/acs....
Unraveling the Molecular Basis of Cooperativity in Cholera Toxin–Glycan Interactions
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.
doi.org
GlycoShape@glycoshape.org · 2mo agoCarbohydrate-binding is a small profit affair, so proteins often adopt complex multidomain architectures enabling a mechanism known as 'binding cooperativity', where binding to one monomer contributes to the binding affinity of the whole systems BUT how does it actually work? 🔥 #glycotime 🧵1/4 ⬇️
