Unprecedented Binding Cooperativity between CuI and CuII in the Copper Resistance Protein CopK from Cupriavidus metallidurans CH34: Implications from Structural Studies by NMR Spectroscopy and X-Ray Crystallography

被引:35
|
作者
Chong, Lee Xin [2 ,3 ]
Ash, Miriam-Rose [5 ]
Maher, Megan J. [1 ]
Hinds, Mark G. [4 ]
Xiao, Zhiguang [2 ,3 ]
Wedd, Anthony G. [2 ,3 ]
机构
[1] Centenary Inst Canc Med & Cell Biol, Newtown, NSW 2042, Australia
[2] Univ Melbourne, Sch Chem & Biomol Sci, Parkville, Vic 3010, Australia
[3] Univ Melbourne, Inst Biotechnol, Parkville, Vic 3010, Australia
[4] Walter & Eliza Hall Inst Med Res, Parkville, Vic 3052, Australia
[5] Univ Sydney, Sch Mol & Microbial Sci, Sydney, NSW 2006, Australia
基金
澳大利亚研究理事会;
关键词
ESCHERICHIA-COLI; INTERMOLECULAR TRANSFER; PSEUDOMONAS-SYRINGAE; TRAFFICKING PROTEIN; TERMINAL DOMAIN; METAL-BINDING; OXIDASE; PLASMID; CU(I); MECHANISMS;
D O I
10.1021/ja807354z
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
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.
引用
收藏
页码:3549 / 3564
页数:16
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