Location and properties of metal-binding sites on the human prion protein - PubMed (original) (raw)

Location and properties of metal-binding sites on the human prion protein

G S Jackson et al. Proc Natl Acad Sci U S A. 2001.

Abstract

Although a functional role in copper binding has been suggested for the prion protein, evidence for binding at affinities characteristic of authentic metal-binding proteins has been lacking. By presentation of copper(II) ions in the presence of the weak chelator glycine, we have now characterized two high-affinity binding sites for divalent transition metals within the human prion protein. One is in the N-terminal octapeptide-repeat segment and has a K(d) for copper(II) of 10(-14) M, with other metals (Ni(2+), Zn(2+), and Mn(2+)) binding three or more orders of magnitude more weakly. However, NMR and fluorescence data reveal a previously unreported second site around histidines 96 and 111, a region of the molecule known to be crucial for prion propagation. The K(d) for copper(II) at this site is 4 x 10(-14) M, whereas nickel(II), zinc(II), and manganese(II) bind 6, 7, and 10 orders of magnitude more weakly, respectively, regardless of whether the protein is in its oxidized alpha-helical (alpha-PrP) or reduced beta-sheet (beta-PrP) conformation. A role for prion protein (PrP) in copper metabolism or transport seems likely and disturbance of this function may be involved in prion-related neurotoxicity.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Metal binding to recombinant human α-PrP91–231. The main figure shows the data for the binding of copper(II) to human PrP91–231 in the presence of increasing concentrations of glycine. The quenching of intrinsic fluorescence from tryptophan-99 is shown as a percentage of the starting fluorescence and is plotted versus the concentration of copper(II) ions. The data in the presence of 100 μM glycine are shown by solid circles, 200 μM by open squares; 500 μM by open circles, and 1 mM by open triangles. The lines superimposed on the data represent an optimal fit of the data to the equation described above. (Inset) A plot of the apparent dissociation constants versus glycine concentration, which clearly vary. Overlaid are the real dissociation constants derived as described above, which are constant with respect to glycine concentration.

Figure 2

Figure 2

NMR signal intensity versus residue number. (A) HSQC peak intensities shown sequentially at 250 μM, 500 μM, 750 μM, and 1000 μM CuSO4 relative to 0 μM CuSO4; showing the effects of addition of Cu2+ to the human PrP91–231. Residues 94–98, 108–114, 135–136, and 153–159 exhibit significant differential alteration of resonances, indicating copper binding. (B) 1H-15N HSQC spectra with 0 μM (a), 375 μM (b), 625 μM (c), and 1000 μM CuSO4 (d), showing the region surrounding peaks arising from H96 and H111. As Cu2+ is titrated into the NMR sample, a general broadening of NMR resonances is observed, due to the presence of Cu2+ in solution. In addition, the peaks arising, for example, from the backbone amide resonances of H96 and H111 exhibit significant further broadening because of their proximity to the binding site of the paramagnetic ion. The peaks arising from the backbone amides of residues N197 and E221 are shown for comparison.

Figure 3

Figure 3

Binding of copper to the octapeptide repeat region PrP52–98. The data for the binding of copper(II) to PrP52–98 in both the presence of 500 μM glycine (open squares) and the absence of glycine (open circles) are shown overlaid. The horizontal dotted line is drawn at a point representing stoichiometry of copper(II) to PrP52–98 (5 μM) to illustrate the loss of the weak-binding site in the presence of glycine. The lines superimposed on the data represent an optimal fit of the data to the equation described above.

Figure 4

Figure 4

Five energetically identical space-filled models of the Cu(II)-binding octapeptide repeat region. Models were generated by calculating the energetic stabilities of all possible combinations of exactly repeating structure in the octapeptide repeat. To enable exhaustive structural evaluation within the repeating unit, a simplified energetic and structural model was applied. Energetic stabilities were calculated by using a united atom model and physicochemical potentials describing hydrophobic, hydrophilic, and backbone hydrogen bonding energies. Conformational space was reduced, by restricting residues to adopt one of six alternative backbone φ–ψ torsion angles. The system is described in detail elsewhere (21). The models presented represent the five most energetically stable conformations of the repeat that also satisfy the proposed coordination of the copper ion (yellow) by the histidine side chains (blue). The remaining side chains are colored gray and backbone atoms are represented by green.

Similar articles

Cited by

References

    1. Prusiner S B. Proc Natl Acad Sci USA. 1998;95:13363–13383. - PMC - PubMed
    1. Jackson G S, Clarke A R. Curr Opin Struct Biol. 2000;10:69–74. - PubMed
    1. Stahl N, Baldwin M A, Teplow D B, Hood L, Gibson B W, Burlingame A L, Prusiner S B. Biochemistry. 1993;32:1991–2002. - PubMed
    1. Pan K-M, Baldwin M A, Nguyen J, Gasset M, Serban A, Groth D, Mehlhorn I, Huang Z, Fletterick R J, Cohen F E, Prusiner S B. Proc Natl Acad Sci USA. 1993;90:10962–10966. - PMC - PubMed
    1. Wadsworth J D F, Hill A F, Joiner S, Jackson G S, Clarke A R, Collinge J. Nat Cell Biol. 1999;1:55–59. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources