Copper binding to the prion protein: Structural implications of four identical cooperative binding sites

Abstract

Evidence is growing to support a functional role for the prion protein (PrP) in copper metabolism. Copper ions appear to bind to the protein in a highly conserved octapeptide repeat region (sequence PHGGGWGQ) near the N terminus. To delineate the site and mode of binding of Cu(II) to the PrP, the copper-binding properties of peptides of varying lengths corresponding to 2-, 3-, and 4-octarepeat sequences have been probed by using various spectroscopic techniques. A two-octarepeat peptide binds a single Cu(II) ion with K d Ϸ 6 M whereas a four-octarepeat peptide cooperatively binds four Cu(II) ions. Circular dichroism spectra indicate a distinctive structuring of the octarepeat region on Cu(II) binding. Visible absorption, visible circular dichroism, and electron spin resonance spectra suggest that the coordination sphere of the copper is identical for 2, 3, or 4 octarepeats, consisting of a square-planar geometry with three nitrogen ligands and one oxygen ligand. Consistent with the pH dependence of Cu(II) binding, proton NMR spectroscopy indicates that the histidine residues in each octarepeat are coordinated to the Cu(II) ion. Our working model for the structure of the complex shows the histidine residues in successive octarepeats bridged between two copper ions, with both the N2 and N␦1 imidazole nitrogen of each histidine residue coordinated and the remaining coordination sites occupied by a backbone amide nitrogen and a water molecule. This arrangement accounts for the cooperative nature of complex formation and for the apparent evolutionary requirement for four octarepeats in the PrP.

The Affinity of Copper Binding to the Prion Protein Octarepeat Domain: Evidence for Negative Cooperativity

“…As shown in our previous work, EPR spectra from a copper titration of PrP (23)(24)(25)(26)(27)(28) show a progression of different spectra and these are assigned to individual binding components (17). Component 3 dominates at low copper occupancy (≤1.0 equiv).…”

“…For instance, equilibrium dialysis measurements performed on PrP(23-98) find a half-maximal binding at 5.9 μM and a Hill coefficient (n) of 3.4, indicating a very strong positive cooperativity through the micromolar range (8). Similar results were obtained from CD measurements of the d-d absorption band at 570 nm, arising from Cu 2+ binding to PrP (58-91) (25). The CD signal increases in a sigmoidal fashion, consistent with positive cooperativity.…”

“…Clearly, the results reported here require not only confirmation in full-length PrP but also an assessment of the influence of non-octarepeat coordination sites (20,28) and other features that may affect Cu 2+ uptake. With regard to octarepeat coordination, however, our previously published experiments on full-length PrP Since the discovery of the physiological connection of PrP to copper, there have been intensive efforts to determine the precise Cu 2+ affinity (8,10,(21)(22)(23)(24)(25). As noted in the Introduction, there is wide disagreement with reported K d values distributed over nearly 8 orders of magnitude.…”

“…For an enzyme, one would expect high affinity, reflected through a low dissociation constant (K d ) (21), whereas buffering or sensing would require a lower affinity, with a K d near the concentration of extracellular copper (22). Unfortunately, investigations into the dissociation constant and the mechanism of copper uptake reveal highly disparate outcomes depending upon the measurement technique and the particular PrP construct (8,10,(21)(22)(23)(24)(25). Current estimates place K d between 10 −6 M (10,22,23) and 10 −14 M (21,24), an experimental uncertainty of 8 orders of magnitude.…”

“…Several reports suggest that the octarepeat domain takes up Cu 2+ with positive cooperativity (8,22,25). For instance, equilibrium dialysis measurements performed on PrP(23-98) find a half-maximal binding at 5.9 μM and a Hill coefficient (n) of 3.4, indicating a very strong positive cooperativity through the micromolar range (8).…”