Copper binding to the prion protein: Structural implications of four identical cooperative binding sites

Evidence is growing to support a functional role for the prion protein (PrP ) in copper metabolism. Copper ions appear to bind to the protein in a high ly conserved octapeptide repeat region (sequence PHGGGWGQ) near the N termi nus. To delineate the site and made of binding of Cu(II) to the PrP, the co pper-binding properties of peptides of varying lengths corresponding to 2-, 3-, and 4-octarepeat sequences have been probed by using various spectrosc opic techniques. A two-octarepeat peptide binds a single Cu(II) ion with K- d approximate to 6 mu M whereas a four-octarepeat peptide cooperatively bin ds four Cu(II) ions. Circular dichroism spectra indicate a distinctive stru cturing of the octarepeat region on Cu(II) binding. Visible absorption, vis ible circular dichroism, and electron spin resonance spectra suggest that t he coordination sphere of the copper is identical for 2, 3, or 4 octarepeat s, consisting of a square-planar geometry with three nitrogen ligands and o ne oxygen ligand. Consistent with the pH dependence of Cu(II) binding, prot on NMR spectroscopy indicates that the histidine residues in each octarepea t are coordinated to the Cu(II) ion. Our working model for the structure of the complex shows the histidine residues in successive octarepeats bridged between two copper ions, with both the N epsilon 2 and N delta 1 imidazole nitrogen of each histidine residue coordinated and the remaining coordinat ion sites occupied by a backbone amide nitrogen and a water molecule. This arrangement accounts for the cooperative nature of complex formation and fo r the apparent evolutionary requirement for four octarepeats in the PrP.