Stability and Cu(II) binding of prion protein variants related to inherited human prion diseases

Abstract

All inherited forms of human prion diseases are linked with mutations in the prion protein (PrP) gene. Here we have investigated the stability and Cu(II) binding properties of three recombinant variants of murine full-length PrP(23-231)-containing destabilizing point mutations that are associated with human Gerstmann-Sträussler-Scheinker disease (F198S), Creutzfeld-Jakob disease (E200K), and fatal familial insomnia (D178N) by electron paramagnetic resonance and circular dichroism spectroscopy. Furthermore, we analyzed the variants H140S, H177S, and H187S of the isolated C-terminal domain of murine PrP, mPrP(121-231), to test a role of the histidine residues in Cu(II) binding. The F198S and E200K variants of PrP(23-231) differed in Cu(II) binding from the wild-type mPrP(23-231). However, circular dichroism spectroscopy indicated that the variants and the wild type did not undergo conformational changes in the presence of Cu(II). The D178N variant showed a high tendency to aggregate at pH 7.4 both with and without Cu(II). At lower pH values, it showed the same Cu(II) binding behavior as the wild type. The analysis allowed for a better location of the Cu(II) binding sites in the C-terminal part of the protein. Our present data indicate that hereditary forms of prion diseases cannot be rationalized on the basis of altered Cu(II) binding or mutation-induced protein destabilization alone.

FIGURE 1

(a) Three-dimensional structure of the folded C-terminal domain of PrP (PrP(121–231)) showing the location of the amino acid residues mutated in the prion protein variants analyzed in this study. (b) Magnification of the histidine 187 environment in the three-dimensional fold of the murine prion protein PrP(121–231).

FIGURE 2

X-band EPR spectra at pH 4. (a) 0.1 mM CuCl₂ in a formic acid·NaOH buffer. (b) 0.1 mM mPrP(23–231) with four molar equivalents of CuCl₂. (c) 0.1 mM of E200K mPrP(23–231) with four molar equivalents of CuCl₂. (d) 0.1 mM of F198S mPrP(23–231) with four molar equivalents of CuCl₂. (e) 0.1 mM of D178N mPrP(23–231) with four molar equivalents of CuCl₂. (f) 0.1 mM of H177S mPrP(121–231) with four molar equivalents of CuCl₂.

FIGURE 3

X-band EPR spectra at pH 6. (a) 0.1 mM CuCl₂ in a sodium cacodylate·HCl buffer. (b) 0.1 mM mPrP(23–231) with four molar equivalents of CuCl₂. (c) 0.1 mM of E200K mPrP(23–231) with four molar equivalents of CuCl₂. (d) 0.1 mM of F198S mPrP(23–231) with four molar equivalents of CuCl₂. (e) 0.1 mM of D178N mPrP(23–231) with four molar equivalents of CuCl₂.

FIGURE 4

X-band EPR spectra at pH 7.4. (a) 0.1 mM CuCl₂ in a MOPS·NaOH buffer. (b) 0.1 mM mPrP(23–231) with four molar equivalents of CuCl₂. (c) 0.1 mM of E200K mPrP(23–231) with four molar equivalents of CuCl₂. (c′) Simulation of (c) using parameters of complex 2 in Table 1. (d) 0.1 mM of F198S mPrP(23–231) with four molar equivalents of CuCl₂. (e) 0.1 mM of H140S mPrP(121–231) with four molar equivalents of CuCl₂. (f) 0.1 mM of H187S mPrP(121–231) with one molar equivalent of CuCl₂.

FIGURE 5

(a) Unfolding transitions of mPrP(23–231) at pH 4 and at an ionic strength of 30 mM (22°C). Protein concentration, 28 μM. mPrP(23–231) wild type (•), variant E200K (▴), variant F198S (▾), and variant D178N (▪). (b) Unfolding transitions of mPrP (23–231) at pH 4 and at an ionic strength of 165 mM (22°C). Protein concentration, 28 μM. mPrP(23–231) wild type (•) and variant E200K (▴). (c) Unfolding transitions of mPrP (23–231) at pH 4 and at an ionic strength of 165 mM (22°C). 14.5 μM mPrP(23–231) wild type (•), 20 μM mPrP(23–231) F198S (▾), and 20 μM mPrP(23–231) D178N (▪).