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

摘要

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 D-2-symmetric, homotetrameric copper-sensitive operon repressor (CsoR) as a function of Cu-I ligation state. Cu-I binding is overall positively cooperative, but is characterized by distinct ligation state-specific cooperativities. Structural transitions occur upon binding the first and fourth Cu-I, 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).