Deconvoluting the Cu2+ binding modes of full-length prion protein.
The prion protein (PrP) is a cell-surface Cu(2+)-binding glycoprotein that when misfolded is responsible for a number of transmissible spongiform encephalopathies. Full-length PrP-(23-231) and constructs in which the octarepeat region has been removed, or His(95) and His(110) is replaced by alanine residues, have been used to elucidate the order and mode of Cu(2+) coordination to PrP-(23-231). We have built on our understanding of the appearance of visible CD spectra and EPR for various PrP fragments to characterize Cu(2+) coordination to full-length PrP. At physiological pH, Cu(2+) initially binds to full-length PrP in the amyloidogenic region between the octarepeats and the structured domain at His(95) and His(110). Only subsequent Cu(2+) ions bind to single histidine residues within the octarepeat region. Ni(2+) ions are used to further probe metal binding and, like Cu(2+), Ni(2+) will bind individually to His(95) and His(110), involving preceding main chain amides. Competitive chelators are used to determine the affinity of the first mole equivalent of Cu(2+) bound to full-length PrP; this approach places the affinity in the nanomolar range. The affinity and number of Cu(2+) binding sites support the suggestion that PrP could act as a sacrificial quencher of free radicals generated by copper redox cycling.
School of Biological and Chemical Sciences, Queen Mary, University of London, Mile End Road, London, E1 4NS, United Kingdom.
Mark
M
Klewpatinond
Paul
P
Davies
Suzanne
S
Bowen
David R
DR
Brown
John H
JH
Viles
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002413:Cations, Divalent / Q000737:chemistry / Q000378:metabolism; D002614:Chelating Agents / Q000737:chemistry; D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D016166:Free Radical Scavengers / Q000737:chemistry* / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D051379:Mice; D009532:Nickel / Q000737:chemistry / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D011328:Prions / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017510:Protein Folding
D002413:Cations, Divalent; D002614:Chelating Agents; D016166:Free Radical Scavengers; D006023:Glycoproteins; D009842:Oligopeptides; D011328:Prions; D003300:Copper; D009532:Nickel