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

HMA2, HMA4, and HMA7 are three of the eight heavy metal transporting P-IB-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 Cut with an affinity between those of HMA4n and HMA7n but binds Zn-II more weakly than either parent protein does. The four MBDs bind C-I more strongly than Zn-II by factors of > 10(6). 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 similar to 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 beta alpha beta beta alpha beta 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 Cd-113 chemical shift in [Cd-113]HMA4n was consistent with a (CdS2OX)-S-II (X = O or N) coordination sphere. The Zn-II form of the Cu-I transporter HMA7n is a monomer in solution but crystallized its 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.