Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. Here, we identify a new copper-specific repressor (CsoR) of a copper-sensitive operon (cso) in Mycobacterium tuberculosis (Mtb) that is representative of a large, previously uncharacterized family of proteins (DUF156). Electronic and X-ray absorption spectroscopies reveal that CsoR binds a single-monomer mole equivalent of Cu(I) to form a trigonally coordinated (S2N) Cu(I) complex. The 2.6-Å crystal structure of copper-loaded CsoR shows a homodimeric antiparallel four-helix bundle architecture that represents a novel DNA-binding fold. The Cu(I) is coordinated by Cys36, Cys65′ and His61′ in a subunit bridging site. Cu(I) binding negatively regulates the binding of CsoR to a DNA fragment encompassing the operator-promoter region of the Mtb cso operon; this results in derepression of the operon in Mtb and the heterologous host Mycobacterium smegmatis. Substitution of Cys36 or His61 with alanine abolishes Cu(I)- and CsoR-dependent regulation in vivo and in vitro. Potential roles of CsoR in Mtb pathogenesis are discussed.
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We thank N. Echols and T. Alber for their help in the X-ray data collection for the selenomethionine-CsoR crystals, and we thank X. Chen for many helpful discussions. This work was supported by grants from the US National Institutes of Health (GM042569 to D.P.G. and AI068135 to J.C.S.), the Robert A. Welch Foundation (A1295 to D.P.G.) and the US Department of Agriculture (WIS04794 and WIS04823 to A.M.T.). S.K.W. was supported by a US National Institutes of Health Molecular Biosciences Training Grant (T32 GM007215). SSRL is funded by the US Department of Energy and the US National Institutes of Health. The CLS is supported by the Natural Sciences and Engineering Research Council of Canada (NSERC), the National Research Council Canada, the Canadian Institutes of Health Research (CIHR) and the University of Saskatchewan. Research at the University of Saskatchewan was supported by the NSERC and the CIHR, a Canada Research Chair award (G.N.G.), and the University of Saskatchewan, the Province of Saskatchewan, and the US National Institutes of Health (GM057375).
The authors declare no competing financial interests.
The rv0967–rv0970 operon is expressed as a polycistronic operon in Mtb. (PDF 1203 kb)
The expression from the cso promoter is specifically induced by addition of copper salts to the growth medium in M. smegmatis. (PDF 361 kb)
Mapping the CsoR-DNA binding fragment in the operator/promoter region of cso operon by EMSA. (PDF 3336 kb)
Two distinct forms of recombinant CsoR can be purified from E. coli using nondenaturing methods and SDS-PAGE analysis of various Mtb CsoR samples. (PDF 7097 kb)
Copper and DNA binding properties of variant apo-CsoRs. (PDF 836 kb)
Crystallographic data collection and refinement statistics for Mtb CsoR. (PDF 75 kb)
EXAFS curve-fitting results. (PDF 75 kb)
Additional details on methods used and physical characterization of CsoR. (PDF 248 kb)
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Liu, T., Ramesh, A., Ma, Z. et al. CsoR is a novel Mycobacterium tuberculosis copper-sensing transcriptional regulator. Nat Chem Biol 3, 60–68 (2007). https://doi.org/10.1038/nchembio844
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