Fragment length influences affinity for Cu2+ and Ni2+ binding to His96 or His111 of the prion protein and spectroscopic evidence for a multiple histidine binding only at low pH.

The prion protein (PrP) is a Cu2+-binding cell-surface glycoprotein. Using various PrP fragments and spectroscopic techniques, we show that two Cu2+ ions bind to a region between residues 90 and 126. This region incorporates the neurotoxic portion of PrP, vital for prion propagation in transmissible spongiform encephalopathies. Pentapeptides PrP-(92-96) and PrP-(107-111) represent the minimum motif for Cu2+ binding to the PrP-(90-126) fragment. Consequently, we were surprised that the appearance of the visible CD spectra for two fragments of PrP, residues 90-126 and 91-115, are very different. We have shown that these differences do not arise from a change in the co-ordination geometry within the two fragments; rather, there is a change in the relative preference for the two binding sites centred at His111 and His96. These preferences are metal-, pH- and chain-length dependent. CD indicates that Cu2+ initially fills the site at His111 within the PrP-(90-126) fragment. The pH-dependence of the Cu2+ co-ordination is studied using EPR, visible CD and absorption spectroscopy. We present evidence that, at low pH (5.5) and sub-stoichiometric amounts of Cu2+, a multiple histidine complex forms, but, at neutral pH, Cu2+ binds to individual histidine residues. We have shown that changes in pH and levels of extracellular Cu2+ will affect the co-ordination mode, which has implications for the affinity, folding and redox properties of Cu-PrP.