β-cleavage of the human prion protein impacts Cu(II) coordination at its non-octarepeat region

The cellular prion protein (PrP^C) is a membrane-anchored copper binding protein that undergoes proteolytic processing. β-cleavage of PrP^C is associated with a pathogenic condition and it yields two fragments: N2 with residues 23-89, and C2 including residues 90-231. The membrane-bound C2 fragment retains the Cu binding sites at His96 and His111, but it also has a free N-terminal NH₂ group. In this study, the impact of β-cleavage of PrP^C in its Cu(II) binding properties was evaluated, using the peptide of the human prion protein hPrP(90-115) as a model for the C2 fragment. The Cu(II) coordination properties of hPrP(90-115) were studied using circular dichroism (CD) and electron paramagnetic resonance (EPR); while the H96A and H111A substitutions and its acetylated variants were also studied. Cu binding to hPrP(90-115) is dependent on metal ion concentration: At low copper concentrations the participation of His96 and free NH₂-terminus is evident, while at high copper concentrations the His111 site is populated without participation of the N-terminal NH₂ group. The presence of a free NH₂-terminal group in the C2 fragment significantly impacts the Cu(II) coordination properties of the His96 site, where the NH₂ group also anchors the metal ion. This study provides further insights into the impact of proteolytic processing of PrP^C in the Cu binding properties of this important neuronal protein.