Molecular Insights into the Metal Selectivity of the Copper(I)-Sensing Repressor CsoR from Bacillus subtilis

Bacillus subtilis CsoR (Bsu CsoR) is a copper-sensing transcriptional repressor that regulates the expression of the copZA operon encoding a copper chaperone and a Cu efflux P-type ATPase, respectively. Bsu CsoR is a homologue of Mycobacterium tuberculosis CsoR (Mtb CsoR), representative of a large Cu(I)-sensing regulatory protein family. We show here that Bsu CsoR binds approximate to 1 mol equiv of Cu(I) per monomer in vitro with an affinity >= 10(21) M-1. X-ray absorption spectroscopy shows Cu(I) adopts a trigonal S2N coordination like Mtb CsoR. Both apo and Cu(I)-bound Bsu CsoR are stable tetramers in the low micromolar monomer concentration range by sedimentation velocity and equilibrium ultracentrifugation. Apo-Bsu CsoR binds to a pseudopalindromic 30 bp copZA operator-promoter DNA with a stoichiometry of two tetramers per DNA and stepwise affinities of K-1(apo) = 3.1(+/- 0.8) x 10(7) M-1 and K-2(apo), = 8.3 (+/- 2.2) x 10(7) M-1 (0.4 M NaCl, 25 degrees C, pH 6.5). Cu(I) Bsu CsoR binds to the same DNA with greatly reduced affinities, K-1(Cu) = 2.9(+/- 0.4) x 10(6) M-1 and K-2(Cu) <= 1.0 x 10(5) M-1 consistent with a copper-dependent derepression model. This Cu-dependent regulation is abrogated by a "second shell" Glu90-to-Ala substitution. Bsu CsoR binds Ni(II) with very high affinity but forms a non-native coordination geometry, as does Co(II) and likely Zn(II); none of these metals strongly regulates copZA operator DNA binding in vitro. The implications of these findings on the specificity of metal-sensing sites in CsoR/RcnR proteins are discussed.