title: The affinity of copper binding to the prion protein octarepeat domain: evidence for negative cooperativity.
abstract: The prion protein (PrP) binds Cu(2+) in its N-terminal octarepeat domain, composed of four or more tandem PHGGGWGQ segments. Previous work from our laboratory demonstrates that copper interacts with the octarepeat domain through three distinct coordination modes at pH 7.4, depending upon the precise ratio of Cu(2+) to protein. Here, we apply both electron paramagnetic resonance (EPR) and fluorescence quenching to determine the copper affinity for each of these modes. At low copper occupancy, which favors multiple His coordination, the octarepeat domain binds Cu(2+) with a dissociation constant of 0.10 (+/-0.08) nM. In contrast, high copper occupancy, involving coordination through deprotonated amide nitrogens, exhibits a weaker affinity characterized by dissociation constants in the range of 7.0-12.0 microM. Decomposition of the EPR spectra reveals the proportions of all coordination species throughout the copper concentration range and identifies significant populations of intermediates, consistent with negative cooperativity. At most copper concentrations, the Hill coefficient is less than 1.0 and approximately 0.7 at half copper occupancy. These findings demonstrate that the octarepeat domain is responsive to a remarkably wide copper concentration range covering approximately 5 orders of magnitude. Consideration of these findings, along with the demonstrated ability of the protein to quench copper redox activity at high occupancy, suggests that PrP may function to protect cells by scavenging excess copper.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, USA.
authors.forename: Eric D
authors.initials: ED
authors.lastname: Walter
authors.forename: Madhuri
authors.initials: M
authors.lastname: Chattopadhyay
authors.forename: Glenn L
authors.initials: GL
authors.lastname: Millhauser
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002942:Circular Dichroism / Q000379:methods; D003300:Copper / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D011328:Prions / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D020449:Repetitive Sequences, Amino Acid; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D011328:Prions; D006639:Histidine; D003300:Copper