Abstract
The prion protein (PrP) is a Cu(2+) binding cell surface glycoprotein that can misfold into a beta-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 Cu(2+) binding to the full-length protein. In contrast to previous reports we show that Cu(2+) binds to this region of PrP with a nanomolar dissociation constant. NMR and EPR spectroscopy indicate a square-planar or square-pyramidal Cu(2+) coordination utilizing histidine residues. Studies with PrP analogues show that the high affinity site requires both His(96) and His(111) as Cu(2+) ligands, rather than a complex centered on His(96) as has been previously suggested. Our circular dichroism studies indicate a loss of irregular structure on copper coordination with an increase in beta-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 Cu(2+) in prion misfolding and disease must now be re-evaluated in the light of these findings.
Highlights
The prion protein (PrP) is a Cu2؉ binding cell surface glycoprotein that can misfold into a -sheet-rich conformation to cause prion diseases
Recent publications on copper binding to the octarepeats include circular dichroism (CD) studies (24), electron paramagnetic resonance spectroscopy (EPR) studies (25), and a crystallographic study (26)
The changes in the CD spectra upon copper coordination indicate an increase in -sheet or extended conformation, accompanied by a loss of irregular structure
Summary
The prion protein (PrP) is a Cu2؉ binding cell surface glycoprotein that can misfold into a -sheet-rich conformation to cause prion diseases. The majority of copper binding studies have concentrated on the octarepeat region of PrP. 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. Whereas normal physiological function of the prion protein is yet to be determined, the ability of PrPC to bind Cu2ϩ in vivo and in vitro suggests a role in copper homeostasis (4, 5). Residues 60 –91 consist of an octapeptide sequence, PHGGGWGQ, which is repeated four times It is this unstructured region that binds four Cu2ϩ ions cooperatively (5). The majority of studies have concentrated on characterizing the four copper ions binding to the octarepeat region of PrP. Dissociation constants for Cu2ϩ binding to the octarepeat region have been reported to be between 1 nM and 10 M (21, 24) others have suggested a much higher affinity with a Kd in the femtomolar range (22)
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