Dynamic view of an allosteric intermediate in a positively cooperative dimer.

Positive cooperativity in ligand binding is a hallmark of allosteric oligomers, yet how the first binding event enhances the second remains obscure, because the pivotal singly-bound intermediate (lig1) is thermodynamically disfavored and rarely accumulates. Distinguishing concerted (MWC) from sequential (KNF) mechanisms turns on one question: when a ligand binds one protomer, does its empty partner change conformation? Here we resolve this for the allosteric homodimer chorismate mutase (CM) using mixed-labeled dimers (MLDs), in which a single maleimide crosslink stabilizes a heterodimer carrying one NMR-labeled and one active-site-inactivated subunit, trapping lig1 for prolonged study. Isothermal titration calorimetry shows that inhibitor binding to CM is positively cooperative and entirely entropy-driven, with the second event carrying far larger enthalpic and entropic swings than the first. Protomer-resolved NMR reveals that the first binding event switches both subunits to the relaxed (R) state-a concerted, MWC-like transition that rules out a strictly sequential model-yet the empty subunit is not a clean R conformer but a "fuzzy", dynamically heterogeneous ensemble, with extensive microsecond-millisecond motion focused at the dimer interface, and a raft of residues surrounding the empty active site. Backbone probes confirm that both critical 11-12 loops adopt their active posture upon first ligand binding. The mismatch between the chemical-shift picture (MWC-like) and the thermodynamics (weighted toward the second event) argues that cooperativity is not encoded by a simple two-state switch, but by activated dynamics that a purely structural model cannot capture.