Molecular Insights into the Metal Selectivity of the Copper(I)-Sensing Repressor CsoR from Bacillus subtilis
- 27 February 2009
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 48 (15), 3325-3334
- https://doi.org/10.1021/bi900115w
Abstract
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 ≈1 mol equiv of Cu(I) per monomer in vitro with an affinity ≥1021 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 K1apo = 3.1(±0.8) × 107 M−1 and K2apo = 8.3 (±2.2) × 107 M−1 (0.4 M NaCl, 25 °C, pH 6.5). Cu(I) Bsu CsoR binds to the same DNA with greatly reduced affinities, K1Cu = 2.9(±0.4) × 106 M−1 and K2Cu ≤ 1.0 × 105 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.Keywords
This publication has 34 references indexed in Scilit:
- A CuI-Sensing ArsR Family Metal Sensor Protein with a Relaxed Metal Selectivity ProfileBiochemistry, 2008
- Identification of a copper-binding metallothionein in pathogenic mycobacteriaNature Chemical Biology, 2008
- Ni(II) and Co(II) Sensing by Escherichia coli RcnRJournal of the American Chemical Society, 2008
- The Global Responses of Mycobacterium tuberculosis to Physiological Levels of CopperJournal of Bacteriology, 2008
- A place for thioether chemistry in cellular copper ion recognition and traffickingNature Chemical Biology, 2008
- Characterization of the CopR Regulon of Lactococcus lactis IL1403Journal of Bacteriology, 2008
- CsoR regulates the copper efflux operon copZA in Bacillus subtilisMicrobiology, 2007
- Nickel-Dependent Oligomerization of the Alpha Subunit of Acetyl-Coenzyme A Synthase/Carbon Monoxide DehydrogenaseBiochemistry, 2007
- Individual Metal Ligands Play Distinct Functional Roles in the Zinc Sensor Staphylococcus aureus CzrAJournal of Molecular Biology, 2006
- Structure and chemistry of the copper chaperone proteinsCurrent Opinion in Chemical Biology, 2000