Abstract
The copper-sensing operon repressor (CsoR) is representative of a major Cu(I)-sensing family of bacterial metalloregulatory proteins that has evolved to prevent cytoplasmic copper toxicity. It is unknown how Cu(I) binding to tetrameric CsoRs mediates transcriptional derepression of copper resistance genes. A phylogenetic analysis of 227 DUF156 protein members, including biochemically or structurally characterized CsoR/RcnR repressors, reveals that Geobacillus thermodenitrificans (Gt) CsoR characterized here is representative of CsoRs from pathogenic bacilli Listeria monocytogenes and Bacillus anthracis. The 2.56 Å structure of Cu(I)-bound Gt CsoR reveals that Cu(I) binding induces a kink in the α2-helix between two conserved copper-ligating residues and folds an N-terminal tail (residues 12-19) over the Cu(I) binding site. NMR studies of Gt CsoR reveal that this tail is flexible in the apo-state with these dynamics quenched upon Cu(I) binding. Small angle x-ray scattering experiments on an N-terminally truncated Gt CsoR (Δ2-10) reveal that the Cu(I)-bound tetramer is hydrodynamically more compact than is the apo-state. The implications of these findings for the allosteric mechanisms of other CsoR/RcnR repressors are discussed.
Highlights
The copper-sensing operon repressor (CsoR) is representative of a large family of poorly understood copper sensors
We used a phylogenetic approach to establish the evolutionary relationships among these characterized DUF156 proteins, with an emphasis on determining the relatedness among Geobacillus thermodenitrificans CsoR (Gt CsoR) [28] and other CsoR-like copper-sensing repressors and to uncover shared features important for Cu(I) sensing
Our crystal structure of a new Cu(I)-sensing CsoR from G. thermodenitrificans differs in important ways from the other known Cu(I)-bound CsoR structure from M. tuberculosis [14], which we show here is only distantly related to other Cu(I)-specific CsoRs
Summary
The copper-sensing operon repressor (CsoR) is representative of a large family of poorly understood copper sensors. The x-ray crystallographic structure of Cu(I)-bound Gt CsoR and companion NMR and small angle x-ray scattering (SAXS) experiments provide new insights into the Cu(I)-dependent conformational switching associated with allosteric negative regulation of DNA binding by Cu(I).
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