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 (PrPC) 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). PrPC is supposed to bind Cu2+ 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 PrPC. 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 dose 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.