DOI: 10.1002/chem.200601225 Structural and Dynamic Characterization of Copper(II) Binding of the Human Prion Protein Outside the Octarepeat Region Francesco Berti, [c] Elena Gaggelli, [b] Remo Guerrini, [d] Anna Janicka, [a] Henryk Kozlowski,* [a] Anna Legowska, [e] Hanna Miecznikowska, [e] Caterina Migliorini, [b] Rebecca Pogni, [b] Maurizio Remelli, [f] Krzysztof Rolka, [e] Daniela Valensin, [b] and Gianni Valensin* [b] Introduction The prion protein, PrP C , is generally agreed to normally play a functional role in copper transport, sequestration, or antioxidant activity. [1] PrP C may undergo a conformational transformation to a protease-resistant isoform, known as PrP Sc , which is prone to aggregation. PrP Sc is the putative in- fectious agent of transmissible spongiform encephalopathies (TSE) or prion diseases. [2] These diseases have been linked to an imbalance of metal homeostasis in the brain and hy- potheses have been made for an association with the loss of copper binding from PrP C on conversion into PrP Sc . [3,4] The two prion isoforms are identical in primary structure, but they differ in secondary-structure elements [5] and possess considerably different physicochemical properties. [6–10] PrP C is a proteinase-K-sensitive a-helical monomer, [11–14] while PrP Sc is an assembled multimer that is resistant to proteinase digestion and has an increased amount of b structure. [15] The ability of the prion protein to bind Cu II in vitro, as well as in vivo, is well documented and widely suggested to play a relevant role in copper homeostasis or in copper- based enzymatic activity. [16–21] Abstract: Human prion protein (hPrP) fragments encompassing the 91–120 region, namely hPrP92–100 (SP1), hPrP106–113 (SP2), hPrP91–120 (LP1), and hPrP91–114 (LP2), were consid- ered for delineation of the Cu II -binding site(s). NMR and EPR spectroscopy results obtained from LP1 or LP2 were compared with those obtained from SP1 and SP2. The coexistence of two binding sites, one centered at His96 and the other at His111, was evidenced and ratified by ESI mass spectrometry at low and high metal:peptide ratios. While room-temperature NMR spec- troscopy data were consistent with the binding site centered on His111 being approximately fourfold stronger than that centered on His96, low-tempera- ture EPR spectroscopy results yielded evidence for the opposite trend. This disagreement, which has also occurred in the literature, was clarified by tem- perature-dependent molecular dynam- ics runs that demonstrated Met112 ap- proaching the metal at room tempera- ture, a process that is expected to stabi- lize the His111-centered binding site through hydrophobic shielding of the metal coordination sphere. Keywords: binding studies · coordi- nation modes · copper · peptides · prion proteins [a] Dr. A. Janicka, Prof. H. Kozlowski Faculty of Chemistry, University of Wroclaw F. Joliot-Curie 14, 50383 Wroclaw (Poland) Fax:(+ 48)71-375-7251 E-mail:henrykoz@wchuwr.chem.uni.wroc.pl. [b] Prof. E. Gaggelli, Dr. C. Migliorini, Prof. R. Pogni, Dr. D. Valensin, Prof. G. Valensin Department of Chemistry, University of Siena via Aldo Moro, 53100 Siena (Italy) Fax:(+ 39)0577-234-231 E-mail:valensin@unisi.it. [c] Dr. F. Berti Novartis Vaccines, 53100 Siena (Italy) [d] Dr. R. Guerrini Department of Pharmaceutical Sciences University of Ferrara, Ferrara (Italy) [e] Dr. A. Legowska, Dr. H. Miecznikowska, Prof. K. Rolka Faculty of Chemistry, University of Gdansk, Gdansk (Poland) [f] Prof. M. Remelli Department of Chemistry, University of Ferrara, Ferrara (Italy) Supporting information for this article is available on the WWW under http://www.chemeurj.org/ or from the author. Chem. Eur. J. 2006, 00,0–0 # 2006 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim These are not the final page numbers! ÞÞ &1& FULL PAPER