CopY as a Transcriptional Duet Orchestrating Copper Homeostasis via Asymmetry-Dependent DNA Affinity

Copper is essential yet potentially toxic, requiring tight regulation of intracellular concentration. The copper-responsive transcriptional repressor CopY is unusual among bacterial metalloregulators because it occupies two operator sites within the cop promoter. Although two CopY dimers bind these operators independently, the mechanistic significance of this dual-operator architecture remains unclear. Here, we combine biochemical assays, circular dichroism (CD), and electron paramagnetic resonance (EPR) distance measurements to define the molecular basis of CopY-mediated repression. CopY binds the proximal operator with higher affinity than the distal operator. CD measurements further show that DNA binding increases CopY thermal stability, with greater stabilization by the proximal operator. Conversely, DNA-based EPR measurements reveal comparable CopY-induced compaction of both operators. Thus, protein stabilization and DNA deformation represent distinct components of repression. Moreover, site-directed spin labeling and EPR demonstrate that the metal binding domain is conformationally heterogeneous and highly flexible. DNA binding has only a marginal effect on this flexibility. Thus, the metal site is not a rigid pocket but a dynamic one, even in the DNA-bound repressor, a property that may facilitate Cu(I) capture and hence derepression once copper levels rise. We propose that the proximal operator serves as a high-affinity anchoring site, whereas the distal operator primarily promotes promoter remodeling, and that the plasticity of the metal binding domain is retained in the DNA-bound repressor. Together, these findings support a "transcriptional duet" model in which two CopY dimers bound to non-equivalent operators make distinct and complementary contributions to repression.