protein surface. The electrostatic potential on a grid point r was calculated by using the classical expression: V ( !r)~ 1 XN 4pe0 j~1 qj !R { !r j where N is the number of protein atoms, and !Rj and qj the vector associated to position of jth protein atom, and its OPLS-AA charge, respectively [44]. The MEP similarity between model I and II was then evaluated at different portion of the grid by the calculation of Carbo´ similarity index (sC) [47] using the following formula: Npoints P VA(i)VB(i) sC ½A,B ~ sﬃNﬃﬃpﬃﬃoﬃiﬃnﬃﬃtﬃsﬃﬃiﬃ~ﬃﬃﬃ1ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃNﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ points P ½VA(i) 2 P ½VB(i) 2 i~1 i~1 where VI(i) and VII(i) are the corresponding MEPs calculated at ith point, and Npoints is the number of grid points considered for the calculation of similarity. Moreover, because the important role played by the H3 helix, characterizing the whole protein frame, we performed our similarity analysis with respect to its axis (approximately taken as the x-axis in the workspace coordinates of Maestro). Accordingly, the grid space was partitioned into slices in the yz plane, i.e. perpendicular to the x axis, and sC was calculated on each slice (defined as a set of 6 layers of points sharing the same x coordinate) by including only the points external to the protein surface. The whole grid space was partitioned into 34 slices and the whole procedure was parallelized thus providing higher computational performance. An estimate of sC as a function of the position of the slice along the x axis was thus obtained including in the previous formula only the points of single yz-slices. Ca2+ Binding to Wild Type PrPC The building of a molecular model for the binding of the Ca++ 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 Arg 156, His187, Thr191 and Glu196 (see Results and Discussion). References 1. Prusiner SB (2001) N Engl J Med 344 (20) 1516–1526. 2. Collinge J (2001) Annu Rev Neurosci 24 519–550. 3. Hill AF, Desbruslais M, Joiner S, Sidle KC, Gowland I, et al. (1997) Nature 389 (6650) 448–450 526. 4. Stewart LA, Rydzewska LH, Keogh GF, Knight RS (2008) Neurology 70 (15) 1272–1281. 5. Prusiner SB (1998) Proc Natl Acad Sci U S A 95 (23) 13363–13383. 6. Van Everbroeck B, Dobbeleir I, De Waele M, De Leenheir E, Lubke U, et al. (2004) Acta neuropathologica 108 (3) 194–200. 7. Corsaro A, Paludi D, Villa V, D’Arrigo C, Chiovitti K, et al. (2006) Int J Immunopathol Pharmacol 19 (2) 339–356. 8. Jackson GS, Hill AF, Joseph C, Hosszu L, Power A, et al. (1999) Biochim Biophys Acta 1431 (1) 1–13. 9. Swietnicki W, Petersen R, Gambetti P, Surewicz WK (1997) J Biol Chem 272 (44) 27517–27520. 10. Villa V, Corsaro A, Thellung S, Paludi D, Chiovitti K, et al. (2006) Ann N Y Acad Sci 1090 276–291. 11. Colby DW, Wain R, Baskakov IV, Legname G, Palmer CG, et al. (2010) PLoS Pathog 6 (1) e1000736. Calcium Dependent Prion Protein Toxic Conformation Model I (see above) was used as a template to generate an initial guess of model III, by using the Maestro graphical interface. Ca++ binding site was generated by moving the side chain of Arg 156 toward the external bulk, using the ‘‘quick torsion’’ tool of Maestro. The Ca++ ion was then inserted in the site left by the Arg156 side chain and suitably placed to bind the imidazolic nitrogen of His 187, the phenolic oxygen of Thr 191, and the backbone carbonyl oxygen of Arg 156 itself. This structure underwent the same computational treatment of models I and II (see above) consisting of local minimization, LLMOD search, and multiple minimization, which eventually led to model III. Model III was then compared with models I and II with the same procedure employed for the comparison between models I and II. After superposition to model I or II, model III underwent graphical inspection and the mapping of MEP by using the same approach employed in the I/II comparison. Carbo´ similarity index was calculated for both I/III and II/III comparisons as a function of the position along the x axis and the results were compared to the corresponding I/II analyses. Supporting Information Figure S1 Scheme of the Perl script algorithm for the mapping of MEP at the protein surface according to the partitioning scheme into slices along the x axis. (PDF) Figure S2 Ribbon view of protein model I (dark green), II (light cyan) and III (orange) after superposition through minimization of Ca RMSD. The grid points (red spheres) corresponding to the two minima of I/II similarity, the hypothesized Ca++ binding site and the mutation site are also displayed. (PDF) Acknowledgments We would like to thank Simona Gornati and Erica Pizzi for technical support. This work was supported by grants from Italian Ministry of University and Research to TF (PRIN 2008 and FIRB-Accordi di Programma 2011) and MM (PRIN 2007). Author Contributions Conceived and designed the experiments: MM TF NR AA. Performed the experiments: SS AT TB LS FC AM AC ST VV MES BM RG. Analyzed the data: SS TB AC AT ST VV LS FC AM. Contributed reagents/ materials/analysis tools: SS TB AC AT ST VV LS FC AM. Wrote the paper: MM TF NR AA. 12. Kim JI, Cali I, Surewicz K, Kong Q, Raymond GJ, et al. (2010) J Biol Chem 285 (19) 14083–14087. 13. 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