Unprecedented Binding Cooperativity between CuI and CuII in the Copper Resistance Protein CopK from Cupriavidus metallidurans CH34: Implications from Structural Studies by

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• 作者：Lee Xin Chong^† ; ; Miriam-Rose Ash^‡ ; ; Megan J. Maher*^§ ; ; Mark G. Hinds* ; Zhiguang Xiao*^† ; ; Anthony G. Wedd^† ;
• 刊名：Journal of the American Chemical Society
• 出版年：2009
• 出版时间：March 18, 2009
• 年：2009
• 卷：131
• 期：10
• 页码：3549-3564
• 全文大小：557K
• 年卷期：v.131,no.10(March 18, 2009)
• ISSN：1520-5126

文摘

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 ≈ 10⁻⁵ M) whose structure was defined by NMR and X-ray crystallography. The individual molecules feature two antiparallel β-sheets arranged in a sandwich-like structure and interact through C-terminal β-strands. It binds Cu^II with low affinity (K_D(Cu^II) > 10⁻⁶ M) but Cu^I with high affinity (K_D(Cu^I) = 2 × 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 × 10⁻¹³ M and K_D(Cu^II) = 3 × 10⁻¹² M, that is, binding of Cu^II increased the affinity for Cu^I by a factor of 10² and binding of Cu^I increased the affinity for Cu^II by a factor of at least 10⁶. Stable forms of both Cu^ICu^II−CopK and Ag^ICu^II−CopK were isolated readily. Consistent with this unprecedented copper binding chemistry, NMR spectroscopy detected three distinct forms: apo-CopK, Cu^I−CopK and Cu^ICu^II−CopK that do not exchange on the NMR time scale. This information provides a valuable guide to the role of CopK in copper resistance.