The configuration of the Cu2+ binding region in full-length human prion protein

The cellular prion protein (PrPC) is a Cu2+ binding protein connected to the outer cell membrane. The molecular features of the Cu2+ binding sites have been investigated and characterized by spectroscopic experiments on PrPC-derived peptides and the recombinant human full-length PrPC (hPrP-[23-231]). The hPrP-[23-231] was loaded with Cu-63 under slightly acidic (pH 6.0) or neutral conditions. The PrPC/Cu2+-complexes were investigated by extended X-ray absorption fine structure (EXAFS), electron paramagnetic resonance (EPR), and electron nuclear double resonance (ENDOR). For comparison, peptides from the copper-binding octarepeat domain were investigated in different environments. Molecular mechanics computations were used to select sterically possible peptide/Cu2+ structures. The simulated EPR, ENDOR, and EXAFS spectra of these structures were compared with our experimental data. For a stoichiometry of two octarepeats per copper the resulting model has a square planar four nitrogen Cu2+ coordination. Two nitrogens belong to imidazole rings of histidine residues. Further ligands are two deprotonated backbone amide nitrogens of the adjacent glycine residues and an axial oxygen of a water molecule. Our complex model differs significantly from those previously obtained for shorter peptides. Sequence context, buffer conditions and stoichiometry of copper show marked influence on the configuration of copper binding to PrPC.