A reassessment of copper (II) binding in the full-length prion protein
Résumé
It has been shown previously that the unfolded N-terminal domain of the prion protein can bind up to six Cu 2+} ions in vitro. This domain contains four tandem repeats of the octapeptide sequence PHGGGWGQ, which alongside the two histidine residues at positions 96 and 111, contribute to its Cu 2+} binding properties. At maximum metal-ion occupancy each Cu 2+} is coordinated by a single imidazole and deprotonated backbone amide groups. However two recent studies of peptides representing the octapeptide repeat region of the protein, have shown that at low Cu 2+} availability an alternative mode of coordination occurs where the metal ion is bound by multiple histidine imidazoles. Both modes of binding are readily populated at pH 7.4, while mild acidification to pH 5.5 selects in favour of the low occupancy, multiple imidazole binding mode. We have used NMR to resolve how Cu 2+} binds to the full-length prion protein under mildly acidic conditions where multiple histidine coordination is dominant. We show that at pH 5.5 the protein binds two Cu 2+} ions and that all six histidines of the unfolded N-terminal domain and the N terminal amine act as ligands. These two sites are of sufficient affinity to be maintained in the presence of millimolar concentrations of competing exogenous histidine. A previously unknown interaction between the N-terminal domain and a site on the C-terminal domain becomes apparent when the protein is loaded with Cu 2+}. Furthermore, the data reveal that sub-stoichiometric quantities of Cu 2+} will cause self-association of the prion protein in vitro suggesting that Cu 2+} may play a role in controlling oligomerisation in vivo.
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