A Deeper Insight in Metal Binding to the hCtr1 N-terminus Fragment: Affinity, Speciation and Binding Mode of Binuclear Cu2+ and Mononuclear Ag+ Complex Species.

Ctr1 regulates copper uptake and its intracellular distribution. The first 14 amino acidsequence of the Ctr1 ectodomain Ctr1(1-14) encompasses the characteristic Amino Terminal Cu2+and Ni2+ binding motif (ATCUN) as well as the bis-His binding motif (His5 and His6). We report acombined thermodynamic and spectroscopic (UV-vis, CD, EPR) study dealing with the formation ofCu2+ homobinuclear complexes with Ctr1(1-14), the percentage of which is not negligible even in thepresence of a small Cu2+ excess and clearly prevails at a M/L ratio of 1.9. Ascorbate fails to reduceCu2+ when bound to the ATCUN motif, while it reduces Cu2+ when bound to the His5-His6 motifinvolved in the formation of binuclear species. The histidine diade characterizes the second bindingsite and is thought to be responsible for ascorbate oxidation. Binding constants and speciation of Ag+complexes with Ctr1(1-14), which are assumed to mimic Cu+interaction with N-terminus of Ctr1(1-14),were also determined. A preliminary immunoblot assay evidences that the anti-Ctr1 extracellularantibody recognizes Ctr1(1-14) in a different way from the longer Ctr1(1-25) that encompasses a secondHis and Met rich domain.