Metal Binding Affinities of Arabidopsis Zinc and Copper Transporters: Selectivities Match the Relative, but Not the Absolute, Affinities of their Amino-Terminal Domains,

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• 作者：Matthias Zimmermann ; Oliver Clarke ; Jacqui M. Gulbis ; David W. Keizer ; Renee S. Jarvis ; Christopher S. Cobbett ; Mark G. Hinds ; Zhiguang Xiao ; Anthony G. Wedd
• 刊名：Biochemistry
• 出版年：2009
• 出版时间：December 15, 2009
• 年：2009
• 卷：48
• 期：49
• 页码：11640-11654
• 全文大小：1219K
• 年卷期：v.48,no.49(December 15, 2009)
• ISSN：1520-4995

文摘

HMA2, HMA4, and HMA7 are three of the eight heavy metal transporting P_1B-type ATPases in the simple plant Arabidopsis thaliana. The first two transport Zn²⁺, and the third transports Cu⁺. Each protein contains soluble N-terminal metal-binding domains (MBDs) that are essential for metal transport. While the MBD of HMA7 features a CxxC sequence motif characteristic of Cu^I binding sites, those of HMA2 and HMA4 contain a CCxxE motif, unique for plant Zn²⁺-ATPases. The three MBDs HMA2n (residues 1−79), HMA4n (residues 1−96), and HMA7n (residues 56−127) and an HMA7/4n chimera were expressed in Escherichia coli. The chimera features the ICCTSE motif from HMA4n inserted in place of the native MTCAAC motif of HMA7n. Binding affinities for Zn^II and Cu^I of each MBD were determined by ligand competition with a number of chromophoric probes. The challenges of using these probes reliably were evaluated, and the relative affinities of the MBDs were verified by independent cross-checks. The affinities of HMA2n and HMA4n for Zn^II are higher than that of HMA7n by a factor of 20−30, but the relative affinities for Cu^I are inverted by a factor of 30−50. These relativities are consistent with their respective roles in metal selection and transportation. Chimera HMA7/4n binds Cu^I with an affinity between those of HMA4n and HMA7n but binds Zn^II more weakly than either parent protein does. The four MBDs bind Cu^I more strongly than Zn^II by factors of >10⁶. It is apparent that the individual MBDs are not able to overcome the large thermodynamic preference for Cu⁺ over Zn²⁺. This information highlights the potential toxicity of Cu⁺ in vivo and why copper sensor proteins are 6 orders of magnitude more sensitive than zinc sensor proteins. Metal speciation must be controlled by multiple factors, including thermodynamics (affinity), kinetics (including protein−protein interactions), and compartmentalization. The structure of Zn^II-bound HMA4n defined by NMR confirmed the predicted ferredoxin βαββαβ fold. A single Zn atom was modeled onto a metal-binding site with protein ligands comprising the two thiolates and the carboxylate of the CCxxE motif. The observed ¹¹³Cd chemical shift in [¹¹³Cd]HMA4n was consistent with a Cd^IIS₂OX (X = O or N) coordination sphere. The Zn^II form of the Cu^I transporter HMA7n is a monomer in solution but crystallized as a polymeric chain [(Zn^II−HMA7n)_m]. Each Zn^II ion occupied a distorted tetrahedral site formed from two Cys ligands of the CxxC motif of one HMA7n molecule and the amino N and carbonyl O atoms of the N-terminal methionine of another.