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
Rights Notice
Refer to copyright notice on published article.


Creation Date: 2009-03-18 12:00:00
Last Modified: 0001-01-01 12:00:00
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