The prion protein (PrP) is a Cu 2؉ binding cell surface glycoprotein. There is increasing evidence that PrP functions as a copper transporter. In addition, strains of prion disease have been linked with copper binding. We present here CD spectroscopic studies of Cu 2؉ binding to various fragments of the octarepeat region of the prion protein. We show that glycine and L-histidine will successfully compete for all Cu 2؉ ions bound to the PrP octapeptide region, suggesting Cu 2؉ coordinates with a lower affinity for PrP than the fM dissociation constant reported previously. We show that each of the octarepeats do not form an isolated Cu 2؉ binding motif but fold up cooperatively within multiple repeats. In addition to the coordinating histidine side chain residues, we show that the glycine residues and the proline within each octarepeat are also necessary to maintain the coordination geometry. The highly conserved octarepeat region in mammals is a hexarepeat in birds that also binds copper but with different coordination geometry. Finally, in contrast to other reports, we show that Mn 2؉ does not bind to the octarepeat region of PrP.

The prion protein (PrP C ) is a copper binding cell surface glycoprotein which when misfolded causes transmissible spongiform encephalopathies. The cooperative binding of Cu 2+ to an unstructured octarepeat sequence within PrP C causes profound folding of this region. The use of NMR to determine the solution structure of the octarepeat region of PrP with Cu 2+ bound has been hampered by the paramagnetic nature of the Cu 2+ ions. Using NMR we have investigated the binding of candidate diamagnetic replacement ions, to the octarepeat region of PrP. We show that Pd 2+ forms diamagnetic complexes with the peptides HGGG, HGGGW and QPHGGGWGQ with 1 : 1 stoichiometry. The 1 H NMR spectra indicate that these peptides are in slow-exchange between free and bound Pd 2+ on the chemical-shift time-scale. We demonstrate that the Pd-peptide complex forms slowly with a time taken to reach half-maximal signal of 3 hours. Other candidate metal ions, Ni 2+ , Pt 2+ and Au 3+ , were investigated but only the Pd 2+ complexes gave resolvable 1 H NMR spectra. We have determined the solution structure of the QPHGGGWGQ-Pd 1 : 1 complex using 71 NOE distance restraints. A backbone RMSD of 0.30 Å was observed over residues 3 to 7 in the final ensemble. The co-ordinating ligands consist of the histidine imidazole side chain Ne, the amide N of the second and third glycines with possibly H 2 O as the fourth ligand. The co-ordination geometry differs markedly from that of the HGGGW-Cu crystal structure. This survey of potential replacement metal ions to Cu 2+ provides insight into the metal specificity and co-ordination chemistry of the metal bound octarepeats.