Resolution of Stepwise Cooperativities of Copper Binding by the Homotetrameric Copper-Sensitive Operon Repressor (CsoR): Impact on Structure and Stability

The cooperativity of ligand binding is central to biological regulation and new approaches are needed to quantify these allosteric relationships. Herein, we exploit a suite of mass spectrometry (MS) experiments to provide novel insights into homotropic Cu‐binding cooperativity, gas‐phase stabilities and conformational ensembles of the D2‐symmetric, homotetrameric copper‐sensitive operon repressor (CsoR) as a function of CuI ligation state. CuI binding is overall positively cooperative, but is characterized by distinct ligation state‐specific cooperativities. Structural transitions occur upon binding the first and fourth CuI, with the latter occurring with significantly higher cooperativity than previous steps; this results in the formation of a holo‐tetramer that is markedly more resistant than apo‐, and partially ligated CsoR tetramers toward surface‐induced dissociation (SID). Die stufenweisen Kooperativitäten der Cu‐Bindung an den homotetrameren kupfersensitiven Operonrepressor (CsoR) wurden durch Massenspektrometrie aufgelöst, wobei das Ausmaß der Kooperativität auf Gasphaseneigenschaften bezogen wurde. Die Holo(Cu4)‐Struktur in der Gasphase bevorzugt einen kompakteren Zustand und ist deutlich stabiler gegen Fragmentierung als Apo‐ oder partiell Cu‐ligierte Spezies.