Probing copper2+ binding to the prion protein using diamagnetic nickel2+ and 1H NMR: the unstructured N terminus facilitates the coordination of six copper2+ ions at physiological concentrations.
The prion protein (PrP) is a Cu2+ binding cell surface glyco-protein. Misfolding of PrP into a beta-sheet rich conformation is associated with transmissible spongiform encephalopathies. Here we use Ni2+ as a diamagnetic probe to further understand Cu2+ binding to PrP. Like Cu2+, Ni2+ preferentially binds to an unstructured region between residues 90 and 126 of PrP, which is a key region for amyloidogenicity and prion propagation. Using both 1H NMR and visible-circular dichroism (CD) spectroscopy, we show that two Ni2+ ions bind to His96 and His111 independently of each other. 1H NMR indicates that both Ni2+ binding sites form square-planar diamagnetic complexes. We have previously shown that Cu2+ forms a paramagnetic square-planar complex in this region, suggesting that Ni2+ could be used as a probe for Cu2+ binding. In addition, competition studies show that two Cu2+ ions can displace Ni2+ from these sites. Upon Ni2+ addition 1H NMR changes in chemical shifts indicate the imidazole ring and amide nitrogen atoms to the N terminus of both His96 and His111 act as coordinating ligands. Use of peptide fragments confirm that PrP(92-96) and PrP(107-111) represent the minimal binding motif for the two Ni2+ binding sites. Analysis of Cu2+ loaded visible-CD spectra show that as with Ni2+, PrP(90-115) binds two Cu2+ ions at His96 and His111 independently of each other. Visible CD studies with PrP(23-231Delta51-90), a construct of PrP(23-231) with the octarepeat region deleted to improve solubility, confirm binding of Ni2+ to His96 and His111 in octarepeat deleted PrP(23-231). The structure of the Cu/Ni complexes is discussed in terms of the implications for prion protein function and disease.
Jones
Christopher E
CE
School of Biological Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, UK.
Klewpatinond
Mark
M
Abdelraheim
Salama R
SR
Brown
David R
DR
Viles
John H
JH
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008280:Magnetics; D051379:Mice; D008958:Models, Molecular; D009532:Nickel / Q000378:metabolism*; D011257:Pregnancy Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
C057074:Plfr protein, mouse; D011257:Pregnancy Proteins; D011994:Recombinant Proteins; D006639:Histidine; D003300:Copper; D009532:Nickel