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 D2-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 is that markedly more resistant than apo-, and partially-ligated CsoR tetramers toward surface-induced dissociation (SID).

Copper Chaperone CupA and Zinc Control CopY Regulation of the Pneumococcal cop Operon

“…S3C , D , and E ). Taken together, the results show that zinc caused the CopY dimer to dimerize into a tetramer, a form consistent with other copper operon repressors ( 30 – 32 ).…”

Mass spectrometry captures off-target drug binding and provides mechanistic insights into the human metalloprotease ZMPSTE24

“…In conclusion, the mass spectrometry approaches that we applied in this study extend earlier studies of metal binding and drugs to soluble protein targets 40–44 and provide direct evidence for the binding of HIV protease inhibitors to the membrane protein ZMPSTE24, with and without zinc binding, and in the presence of detergent and lipid adducts. This is not only the first direct evidence of drug binding to ZMPSTE24 but our results are corroborated by the resistance to unfolding conferred by these binding events.…”

Metal-dependent allosteric activation and inhibition on the same molecular scaffold: the copper sensor CopY from Streptococcus pneumoniae

“…9B and S6 † ) for the apo-, Zn 1 - and Cu 2 complexed forms of CopY that collectively provide insights into monomer stability and mobility profiles of these allosteric states, using methods analogous to those previously applied to the Cu-sensing repressor CsoR. 44 …”