Authors
Sapienza, P. J., Mileur, T. R., Khan, M. S., Li, K., Aube, J., Lee, A.
Abstract
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 (lig) 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 lig 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 mode--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.
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bioRxiv
The authors list and abstract were imported from bioRxiv on 18 Aug 2026.
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