{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,8,21]],"date-time":"2026-08-21T12:37:02Z","timestamp":1787315822621,"version":"build-2736575974"},"posted":{"date-parts":[[2026,8,17]]},"group-title":"Biophysics","reference-count":63,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T00:00:00Z","timestamp":1786924800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000057","name":"NIGMS","doi-asserted-by":"crossref","award":["GM127698"],"award-info":[{"award-number":["GM127698"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000057","name":"NIGMS","doi-asserted-by":"crossref","award":["GM144348"],"award-info":[{"award-number":["GM144348"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,8,17]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>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\u2081) 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\u2081 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\u2014a concerted, MWC-like transition that rules out a strictly sequential model\u2014yet the empty subunit is not a clean R conformer but a \u201cfuzzy\u201d, dynamically heterogeneous ensemble, with extensive microsecond\u2013 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.<\/jats:p>\n                <jats:sec>\n                  <jats:title>Significance Statement<\/jats:title>\n                  <jats:p>Cooperative ligand binding underlies allosteric control across biology, but mechanisms have been hard to pin down because the crucial half-bound intermediate of a cooperative dimer barely exists at equilibrium. We stabilize this intermediate in the enzyme chorismate mutase by chemically linking one NMR-visible subunit to one that cannot bind ligand, letting us watch each subunit independently. Binding the first ligand flips both subunits to the active state, yet the empty subunit becomes highly dynamic rather than rigidly active. Combined with calorimetry, this shows that the free energy of cooperativity must be stored in ensemble dynamics, not merely in a switch between two structures. The linkage method further opens the door to additional solution-based studies required to better understand allostery and benchmark tools of the future.<\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.08.11.744235","type":"posted-content","created":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T22:00:20Z","timestamp":1787004020000},"source":"Crossref","is-referenced-by-count":0,"title":["Dynamic view of an allosteric intermediate in a positively cooperative dimer"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5279-3653","authenticated-orcid":false,"given":"Paul J.","family":"Sapienza","sequence":"first","affiliation":[{"name":"Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27516, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4538-9764","authenticated-orcid":false,"given":"Trevor R.","family":"Mileur","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Biophysics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Muhammad S.","family":"Khan","sequence":"additional","affiliation":[{"name":"Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27516, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8630-2928","authenticated-orcid":false,"given":"Kelin","family":"Li","sequence":"additional","affiliation":[{"name":"Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27516, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1049-5767","authenticated-orcid":false,"given":"Jeffrey","family":"Aub\u00e9","sequence":"additional","affiliation":[{"name":"Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27516, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2783-1907","authenticated-orcid":false,"given":"Andrew L.","family":"Lee","sequence":"additional","affiliation":[{"name":"Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27516, United States"},{"name":"Department of Biochemistry and Biophysics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026082105100891000_2026.08.11.744235v1.1","doi-asserted-by":"publisher","DOI":"10.1016\/j.tibs.2008.05.009"},{"key":"2026082105100891000_2026.08.11.744235v1.2","unstructured":"M. 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