Calcium Binding Promotes Prion Protein Fragment 90– 231 Conformational Change toward a Membrane Destabilizing and Cytotoxic Structure Sacha Sorrentino1., Tonino Bucciarelli4., Alessandro Corsaro2, Alessio Tosatto1, Stefano Thellung 2 , Valentina Villa2, M. Eugenia Schinina` 3, Bruno Maras3, Roberta Galeno6, Luca Scotti4, Francesco Creati5, Alessandro Marrone5, Nazzareno Re5, Antonio Aceto4, Tullio Florio2", Michele Mazzanti1"* 1 Dipartimento di Bioscienze, University of Milan, Milan, Italy, 2 Sezione di Farmacologia, Dipartimento di Medicina Interna and Centro di Eccellenza per la Ricerca Biomedica (CEBR), University of Genova, Genova, Italy, 3 Dipartimento di Scienze Biochimiche, ‘‘Sapienza’’ University of Rome, Rome, Italy, 4 Dipartimento di Scienze Biomediche, Universita` ‘‘G. d’Annunzio’’ di Chieti-Pescara, Chieti, Italy, 5 Dipartimento di Scienze del Farmaco, Universita` ‘‘G. d’Annunzio’’ di Chieti-Pescara, Chieti, Italy, 6 Istituto Superiore di Sanita`, Roma, Italy Abstract The pathological form of prion protein (PrPSc), as other amyloidogenic proteins, causes a marked increase of membrane permeability. PrPSc extracted from infected Syrian hamster brains induces a considerable change in membrane ionic conductance, although the contribution of this interaction to the molecular mechanism of neurodegeneration process is still controversial. We previously showed that the human PrP fragment 90–231 (hPrP90–231) increases ionic conductance across artificial lipid bilayer, in a calcium-dependent manner, producing an alteration similar to that observed for PrPSc. In the present study we demonstrate that hPrP90–231, pre-incubated with 10 mM Ca++ and then re-suspended in physiological external solution increases not only membrane conductance but neurotoxicity as well. Furthermore we show the existence of a direct link between these two effects as demonstrated by a highly statistically significant correlation in several experimental conditions. A similar correlation between increased membrane conductance and cell degeneration has been observed assaying hPrP90–231 bearing pathogenic mutations (D202N and E200K). We also report that Ca++ binding to hPrP90–231 induces a conformational change based on an alteration of secondary structure characterized by loss of alpha- helix content causing hydrophobic amino acid exposure and proteinase K resistance. These features, either acquired after controlled thermal denaturation or induced by D202N and E200K mutations were previously identified as responsible for hPrP90–231 cytotoxicity. Finally, by in silico structural analysis, we propose that Ca++ binding to hPrP90–231 modifies amino acid orientation, in the same way induced by E200K mutation, thus suggesting a pathway for the structural alterations responsible of PrP neurotoxicity. Citation: Sorrentino S, Bucciarelli T, Corsaro A, Tosatto A, Thellung S, et al. (2012) Calcium Binding Promotes Prion Protein Fragment 90–231 Conformational Change toward a Membrane Destabilizing and Cytotoxic Structure. PLoS ONE 7(7): e38314. doi:10.1371/journal.pone.0038314 Editor: Ilia V. Baskakov, University of Maryland, United States of America Received January 28, 2012; Accepted May 8, 2012; Published July 11, 2012 Copyright: 2012 Sorrentino et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding was provided by the Italian Ministry of University and Research: PRIN 2007 to Mazzanti, PRIN 2007 to Schinina`, PRIN 2008 to Florio, FIRB 2011 to Florio. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: michele.mazzanti@unimi.it . These authors contributed equally to this work. " These authors are joint senior authors on this work. Introduction Prion diseases, also called transmissible spongiform encepha- lopathy (TSE), are fatal neurodegenerative disorders of humans and animals characterized by sporadic, inherited and infective (transmissible) aetiology, including, among the human diseases, Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease, fatal familial insomnia and kuru [1,2]. The epidemic nature of TSE in domestic and wild animals constitutes a serious health problem also for humans. In fact, in past years the appearance of a new variant of CJD, associated to the consumption of bovine spongiform encephalopathy-contaminated beef, created a troubling new scenario in the transmission of prion diseases [3]. TSE are invariably fatal, with death occurring frequently in less than 1 year after the first symptoms appear [4]. From a neuropathology point of view TSE are characterized by cerebral spongiform degeneration, loss of neurons and gliosis, often associated with amyloid deposition [1]. According to the ‘‘protein only’’ hypothesis [5], TSE share a common pathogenic event: the posttranslational misfolding of a membrane-anchored glycoprotein (cellular prion protein, PrPC) into a protease-resistant, aggregation-prone isoform (PrPSc). PrPSc is considered the main, if not the solely, component of the prion, the infectious entity of TSE [1]. PrPC-PrPSc conversion, physiologically prevented by energy barrier, can occur as a spontaneous stochastic event, possibly favored by mutations in the PrP gene (PRNP) or acquired by infection with exogenous PrPSc molecules. The marked resistance to proteolysis of PrPSc, generating fragments of 27–30 KDa (from which the name of PrP27–30), determines its deposition, as partially cleaved protein, in the extracellular space and the formation of amyloid plaques. Neuronal rarefaction and gliosis occur, although not invariantly, in brain areas where a significant PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e38314