Probing copper2+ binding to the prion protein using diamagnetic nickel2+ and 1H NMR : the unstructured N terminus facilitates the coordination of six Cu2+ ions at physiological concentrations

The prion protein (PrP) is a Cu(2+) binding cell surface glyco-protein. Misfolding of PrP into a β-sheet rich conformation is associated with transmissible spongiform encephalopathies. Here we use Ni(2+) as a diamagnetic probe to further understand Cu(2+) binding to PrP. Like Cu(2+), Ni(2+) preferentially binds to an unstructured region between residues 90 and 126 of PrP, which is a key region for amyloidogenicity and prion propagation. Using both 1H NMR and visible-circular dichroism (CD) spectroscopy, we show that two Ni(2+) ions bind to His96 and His111 independently of each other. 1H NMR indicates that both Ni(2+) binding sites form square-planar diamagnetic complexes. We have previously shown that Cu(2+) forms a paramagnetic square-planar complex in this region, suggesting that Ni(2+) could be used as a probe for Cu(2+) binding. In addition, competition studies show that two Cu(2+) ions can displace Ni(2+) from these sites. Upon Ni(2+) addition 1H NMR changes in chemical shifts indicate the imidazole ring and amide nitrogen atoms to the N terminus of both His96 and His111 act as coordinating ligands. Use of peptide fragments confirm that PrP(92-96) and PrP(107-111) represent the minimal binding motif for the two Ni(2+) binding sites. Analysis of Cu(2+) loaded visible-CD spectra show that as with Ni(2+), PrP(90-115) binds two Cu(2+) ions at His96 and His111 independently of each other. Visible CD studies with PrP(23-231Δ51-90), a construct of PrP(23-231) with the octarepeat region deleted to improve solubility, confirm binding of Ni(2+) to His96 and His111 in octarepeat deleted PrP(23-231). The structure of the Cu/Ni complexes is discussed in terms of the implications for prion protein function and disease