Unprecedented Binding Cooperativity between Cu-I and Cu-II in the Copper Resistance Protein CopK from Cupriavidus metallidurans CH34: Implications from Structural Studies by NMR Spectroscopy and X-Ray Crystallography
- Details
- Publication Year 2009-03-18,Volume 131,Issue #10,Page 3549-3564
- Journal Title
- JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
- Publication Type
- Journal Article
- Abstract
- The bacterium Cupriavidus metallidurans CH34 is resistant to high environmental concentrations of many metal ions, including copper. This ability arises primarily from the presence of a large plasmid pMOL30 which includes a cluster of 19 cop genes that respond to copper. One of the protein products CopK is induced at high levels and is expressed to the periplasm as a small soluble protein (8.3 kDa). Apo-CopK associates in solution to form a dimer (K-D approximate to 10(-5) M) whose structure was defined by NMR and X-ray crystallography. The individual molecules feature two antiparallel beta-sheets arranged in a sandwich-like structure and interact through C-terminal P-strands. It binds Cull with low affinity (K-D(Cu-II) > 10(-6) M) but Cu-I with high affinity (K-D(Cu-I) = 2 x 10(-11) M). Cu-I-CopK was also a dimer in the solid state and featured a distorted tetrahedral site Cu-I(S-Met)(3)(NCS). The isothiocyanato ligand originated from the crystallization solution. Binding of Cu-I or Ag-I, but not of Cu-II, favored the monomeric form in solution. While Ag-I-CopK was stable as isolated, Cu-I-CopK was moderately air-sensitive due to a strong binding cooperativity, between Cu-I and Cu-II. This was documented by determination of the Cu-I and Cu-II binding affinities in the presence of the other ion: K-D(Cu-I) = 2 x 10(-13) M and K-D(Cu-II) = 3 x 10(-12) M, that is, binding of Cu-II increased the affinity for Cu-I by a factor of similar to 10(2) and binding of Cu-I increased the affinity for Cu-II by a factor of at least 10(6). Stable forms of both (CuCuII)-Cu-I-CopK and (AgCuII)-Cu-I-CopK were isolated readily. Consistent with this unprecedented copper binding chemistry, NMR spectroscopy detected three distinct forms: apo-CopK, Cu-I-CopK and (CuCuII)-Cu-I-CopK that do not exchange on the NMR time scale. This information provides a valuable guide to the role of CopK in copper resistance.
- Publisher
- AMER CHEMICAL SOC
- Keywords
- ESCHERICHIA-COLI; PSEUDOMONAS-SYRINGAE; INTERMOLECULAR TRANSFER; TRAFFICKING PROTEIN; TERMINAL DOMAIN; DETERMINANT PCO; METAL-BINDING; OXIDASE; PLASMID; CU(I)
- Publisher's Version
- https://doi.org/10.1021/ja807354z
- Terms of Use/Rights Notice
- Refer to copyright notice on published article.
Creation Date: 2009-03-18 12:00:00