slice and the MEP similarity was monitored as a function of their position along the x axis. The plot of the I/II similarity index, reported in Figure 7, shows two minima at 22–23 A˚ and at 62–63 A˚ along x axis, at the beginning and close to the C terminus of H3, respectively (see Figure 11 and S2). The first minima is due to the completely different orientation of the charged Glu168 and the Tyr169 residues in the highly flexible loop 167–171 which significantly differs in the two protein forms. More importantly, the lowest minimum at 62–63 A˚, characterized by a negative value of –0.25 of the Carbo´ index (indicating a large MEP similarity) encompass the region around the protein surface around H1, the C terminus of H2 and the beginning of H3, and comprising the residue at position 200 (Fig. S2). This region also includes the Arg156, His187 and Thr191 residues involved in the H-bond pattern connecting the C terminus of H1 and H2 in the w.t. protein (model I) and switched off in the E200K mutant (model II). The presence of a deep I/II similarity minimum in this region indicates that significant changes in the charge distribution has occurred therein as a consequence of E200K mutation, and is not surprising because of the charge inversion associated to the E200K mutation and the breaking of the H-bond pattern connecting H1 and H2. The graphical inspection of the protein in this region revealed a significant shift of Arg156 as a consequence of E200K mutation: indeed, while in model I the side chain of this residues is directed toward the protein interior, in model II it protrudes outside the protein surface and this shift could contribute to the negative values of I/II similarity index around 62–63 A˚. Binding of Ca++ to the Wild Type PrPC The building of a molecular model for the binding of the Ca2+ ions to the PrPC wild type protein (model III) was carried out on the basis of an inspection of the MEP and of the distribution of potentially coordinating side chains around the w.t. protein surface, suggesting a plausible metal binding site in the close proximity of His187, Thr191 and Glu196. Although His187 and Calcium Dependent Prion Protein Toxic Conformation Thr191 are involved in an H bond pattern with Arg156, that also contributes to keep them in close proximity (Fig. 10, left), this pattern can be easily broken, as shown by the analysis of the E200K mutation above. We thus hypothesized that Arg156 guanidine could shift from its position in the w.t. PrPC allowing the Ca2+ ion to coordinate the His187 and Thr191 and the nearby Glu196. The structure of the resulting PrPC-Ca2+ complex (model III) was calculated by using the same computational approach employed for the calculation of models I and II. The Ca2+ coordination site resulting by our computational approach involved His187, Thr191, Glu196 (g2 coordination) and the backbone carbonyl oxygen of Arg 156 arranged in an distorted square-planar coordination (Fig. 10, right). The effects of Ca2+ coordination on the electrostatic properties of PrPC were evaluated by the analysis of the MEP similarity of model III with models I and II using the same approach described above. The most important feature of the analysis of the I/III MEP similarity as a function of their position along the x axis is the presence of a negative minimum at 62–63 A˚, with the same position and similarity values of the minimum observed for the I/ II similarity (Fig. 11). This result clearly indicates that, the effect of Ca2+ coordination is very similar to that induced by the E200K mutation, leading to a redistribution of the surface charges with patches of positive potential around the beginning of H3. This conclusion was further supports by the analysis of the of II/ III MEP similarity which showed relatively high similarity values (higher than 0.50) at 60–65 A˚ along the x axis, indicating similar electrostatic properties of models II and model III at the beginning of H3. Discussion PrP misfolding is the pathogenic event responsible of the clinical and pathological features of TSE, including neurotoxicity and transmissibility [51]. To date, the exact mechanisms by which the transition PrPCRPrPSc may occur, as well as the final conforma- tion providing PrPSc with gain of toxicity have not been Figure 11. Plot of the Carb o´ similarity index (sC) for the MEPs of models I-III as a function of the position along the x axis. doi:10.1371/journal.pone.0038314.g011 PLoS ONE | www.plosone.org 10 July 2012 | Volume 7 | Issue 7 | e38314