Preferential Cu2+ coordination by His96 and His 111 induces β-sheet formation in the unstructured amyloiodogenic region of the prion protein

Abstract

The prion protein (PrP) is a Cu2+ binding cell surface glycoprotein that can misfold into a β-sheet-rich conformation to cause prion diseases. The majority of copper binding studies have concentrated on the octarepeat region of PrP. However, using a range of spectroscopic techniques, we show that copper binds preferentially to an unstructured region of PrP between residues 90 and 115, outside of the octarepeat domain. Comparison of recombinant PrP with PrP-(91-115) indicates that this prion fragment is a good model for Cu2+ binding to the full-length protein. In contrast to previous reports we show that Cu2+ binds to this region of PrP with a nanomolar dissociation constant. NMR and EPR spectroscopy indicate a square-planar or square-pyramidal Cu2+ coordination utilizing histidine residues. Studies with PrP analogues show that the high affinity site requires both His96 and His111 as Cu2+ ligands, rather than a complex centered on His96 as has been previously suggested. Our circular dichroism studies indicate a loss of irregular structure on copper coordination with an increase in β-sheet conformation. It has been shown that this unstructured region, between residues 90 and 120, is vital for prion propagation and different strains of prion disease have been linked with copper binding. The role of Cu2+ in prion misfolding and disease must now be re-evaluated in the light of these findings.