Brain S100A5 is a novel calcium-, zinc-, and copper ion-binding protein ofthe EF-hand superfamily

S100A5 is a novel member of the EF-hand superfamily of calcium-binding prot eins that is poorly characterized at the protein level. Immunohistochemical analysis demonstrates that it is expressed in very restricted regions of t he adult brain. Here we characterized the human recombinant S100A5, especia lly its interaction with Ca2+, Zn2+, and Cu2+. Flow dialysis revealed that the homodimeric S100A5 binds four Ca2+ ions with strong positive cooperativ ity and an affinity 20-100-fold higher than the other S100 proteins studied under identical conditions. S100A5 also binds two Zn2+ ions and four Cu2ions per dimer, Cu2+ binding strongly impairs the binding of Ca2+; however, none of these ions change the alpha-helical-rich secondary structure. Afte r covalent labeling of an exposed thiol with 2-(4'-(iodoacetamide) anilino) -naphthalene-6-sulfonic acid, binding of Cu2+, but not of Ca2+ or Zn2+, str ongly decreased its fluorescence. In light of the three-dimensional structu re of S100 proteins, our data suggest that in each subunit the single Zn2site 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.