Identification of the Cu2+ binding sites in the N-terminal domain of the prion protein by EPR and CD spectroscopy

Recent evidence indicates that the prion protein (PrP) plays a role in copp er metabolism in the central nervous system. The N-terminal region of human PrP contains four sequential copies of the highly conserved octarepeat seq uence PHGGGWGQ spanning residues 60-91, This region selectively binds dival ent copper ions (Cu2+) in vivo. To elucidate the specific mode and site of binding, we have studied a series of Cu2+-peptide complexes composed of 1-, 2-, and 4-octarepeats and several sub-octarepeat peptides, by electron par amagnetic resonance (EPR, conventional X-band and low-frequency S-band) and circular dichroism (CD) spectroscopy, At pH 7.45, two EPR active binding m odes are observed where the dominant mode appears to involve coordination o f three nitrogens and one oxygen to the copper ion, while in the minor mode two nitrogens and two oxygens coordinate. ESEEM spectra demonstrate that t he histidine imidazole contributes one of these nitrogens. The truncated se quence HGGGW gives EPR and CD that are indistinguishable from the dominant binding mode observed for the multi-octarepeat sequences and may therefore comprise the fundamental Cu2+ binding unit. Both EPR and CD titration exper iments demonstrate rigorously a 1:1 Cu2+/octarepeat binding stoichiometry r egardless of the number of octarepeats in a given peptide sequence. Detaile d spin integration of the EPR signals demonstrates that all of the bound Cu 2+ is detected thereby ruling out strong exchange coupling that is often fo und when there is imidazolate bridging between paramagnetic metal centers. A model consistent with these data is proposed in which Cu2+ is bound to th e nitrogen of the histidine imidazole side chain and to two nitrogens from sequential glycine backbone amides.