A comparative study on the possible zinc binding sites of the human ZnT3 zinc transporter protein

The brain specific zinc transporter protein ZnT3 can be related to the amyloid neuropathology of Alzheimer's disease. In order to analyze the metal binding ability of human ZnT3 protein, here we report a potentiometric and solution structural (UV-Vis, CD, EPR, NMR) study of nickel(II), copper(II) and zinc(II) complexes of three peptides mimicking the possible metal binding sequences of this protein. The peptide L¹ (Ac-RHQAGPPHSHR-NH2) is a minimalist, the cyclic peptide L² (cyclo(Ac-CKLHQAGPPHSHGSRGAEYAPLEEGPEEKC-NH2) is a more complete model of the intracellular His-rich loop, which is widely accepted as a putative metal binding site. The peptide L³ (Ac-PFHHCHRD-NH2) is the model of the conserved cytoplasmic N-terminal -HHCH- sequence. In the physiological pH-range, the ZnL¹, ZnH3L² and ZnL³ complexes are the major species in the corresponding binary systems, with {3N(im)}, {3N(im),2/3O(amide)} and {3N(im),S(-)} coordination environments, respectively. The species ZnL³ has 3-4 orders of magnitude higher stability than the other two complexes, indicating the presence of a high-affinity zinc-binding site at the N-terminal tail of the human ZnT3 transporter. Moreover, L³ shows preferred zinc binding as compared to nickel (log β(ZnL³) - log β(NiL³) = 2.3), probably due to the higher preference of zinc(II) for tetrahedral geometry. These facts suggest that zinc binding to the N-terminal -HHCH- sequence of human ZnT3 may be involved in the biological activity of this zinc transporter protein in zinc sensing, binding or translocation processes.