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 P1B-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 CuI 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 ZnII and CuI 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 ZnII are higher than that of HMA7n by a factor of 20−30, but the relative affinities for CuI 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 CuI with an affinity between those of HMA4n and HMA7n but binds ZnII more weakly than either parent protein does. The four MBDs bind CuI more strongly than ZnII 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 ZnII-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 CdIIS2OX (X = O or N) coordination sphere. The ZnII form of the CuI transporter HMA7n is a monomer in solution but crystallized as a polymeric chain [(ZnII−HMA7n)m]. Each ZnII 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.