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.