Unraveling the Cu²⁺ Binding Sites in the C-Terminal Domain of the Murine Prion Protein:  A Pulse EPR and ENDOR Study

The mammalian prion protein (PrP C ) is a cell surface protein consisting of a flexibly disordered N-terminal segment (residues 23−120) and a structured C-terminal domain (residues 121−231). PrP C is supposed to bind Cu 2+ in vivo, and several studies have recently focused on the ability of this protein to bind divalent cations. In a previous continuous wave electron paramagnetic resonance (CW EPR) study, we showed that Cu(II) binds both to the N- and C-terminal parts of PrP C . Here we present a pulse EPR and electron nuclear double resonance (ENDOR) study of the three different Cu(II) binding sites observed in the structured, C-terminal part of the murine prion protein, mPrP(121−231). It was found that the three complexes are distinguished by a different number of nitrogen atoms directly involved in the Cu(II) ligation. For one of the Cu(II) binding sites that is observed at low pH (3−6), no directly coupled nitrogens could be observed. For a second type of Cu(II) complex, observed at pH 3−8, Davies-ENDOR and hyperfine sublevel correlation (HYSCORE) spectroscopy revealed that histidine is one of the binding ligands. Furthermore, the presence of a nonexchangeable proton close to a copper ion could be demonstrated in a sample containing mainly the second Cu(II) complex. For the third mode of Cu(II) complexation, which can be detected at pH 7−8, Davies-ENDOR spectra indicate that more than one nitrogen atom is directly bound to the copper ion. The observed EPR parameters suggest the involvement of backbone nitrogens in this copper(II) complex.