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

Abstract

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-** This research was supported by the NIH (grant R01 GM042569 to D.P.G.).Correspondence to: David P. Giedroc, giedroc@indiana.edu. Supporting Information for this article, including a complete description of the experimental procedures, is available on the WWW under http://dx.doi.org/10.1002/anie.2015xxxxxWorking Together: The step-wise cooperativities of Cu binding to the homotetrameric copper sensitive operon repressor were resolved by mass spectrometry, with the extent of cooperativity related to gas phase properties. The gas phase holo (Cu 4 ) structure was found to favor a more compact state, and was markedly more resistant to fragmentation than apo-or partially Cu-ligated species.

Copper resistance and its regulation in the sulfate-reducing bacterium Desulfosporosinus sp. OT

“…In the absence of copper, two CsoR tetramers clutch the operator region to prevent transcription On: Fri, 11 May 2018 06:47:07 (Chaplin et al, 2015). For induction of transcription by copper, four Cu + per homotetramer bind cooperatively to a C and an H residue on one subunit and a C residue of the adjacent subunit (Jacobs et al, 2015). This allosterically reduces or inhibits DNA binding, allowing transcription of the downstream genes to proceed .…”

“…S2) (Liu et al, 2007;Chang et al, 2014;Jacobs et al, 2015;Porto et al, 2015). The subunit composition of purified CopU (Fig.…”