Brain S100A5 Is a Novel Calcium-, Zinc-, and Copper Ion-binding Protein of the EF-hand Superfamily

S100A5 is a novel member of the EF-hand superfamily of calcium-binding proteins that is poorly characterized at the protein level. Immunohistochemical analysis demonstrates that it is expressed in very restricted regions of the adult brain. Here we characterized the human recombinant S100A5, especially its interaction with Ca2+, Zn2+, and Cu2+. Flow dialysis revealed that the homodimeric S100A5 binds four Ca2+ ions with strong positive cooperativity and an affinity 20–100-fold higher than the other S100 proteins studied under identical conditions. S100A5 also binds two Zn2+ ions and four Cu2+ ions per dimer. Cu2+ binding strongly impairs the binding of Ca2+; however, none of these ions change the α-helical-rich secondary structure. After covalent labeling of an exposed thiol with 2-(4′-(iodoacetamide)anilino)-naphthalene-6-sulfonic acid, binding of Cu2+, but not of Ca2+ or Zn2+, strongly decreased its fluorescence. In light of the three-dimensional structure of S100 proteins, our data suggest that in each subunit the single Zn2+ site is located at the opposite side of the EF-hands. The two Cu2+-binding sites likely share ligands of the EF-hands. The potential role of S100A5 in copper homeostasis is discussed.