An NMR and Molecular Dynamics investigation of the Avian Prion

The prion protein is a copper binding glycoprotein that in mammals can misfold into a pathogenic isoform leading to prion diseases, as opposed, surprisingly, to avians. The avian prion N-terminal tandem repeat is richer in prolines than the mammal one, and understanding their effect on conformation is of great biological importance. Here we succeeded in investigating the conformations of a single avian hexarepeat by means of NMR and Molecular Dynamics techniques. We found a high flexibility and a strong conformational dependence on pH: local turns are present at acidic and neutral pH, while unordered regions dominate at basic conditions. Introduction The prion protein (PrP) is a cell surface glycosyl-phosphatidylinositol (GPI) anchored glycoprotein [1,2], whose biological function has not yet been identified, even if it has been established that in mammals its conformational change gives rise to the β-sheet rich and pathogenic isoform PrP 1 Corresponding author: prof. Claudio Zannoni, Dipartimento di Chimica Fisica e Inorganica,viale Risorgimento 4, I-40136 Bologna (Italy); fax: +39 051 644702; e-mail: [email protected]. 2 [1,2]. Avian species also express prion protein, but no evidence of neurodegenerative disorders have been reported among them [3-5] and chicken prion protein (ChPrP) has initially been described to induce acetylcholine-receptor activity [4]. This protein shares about 33% of primary sequence identity with the mammalian one and some essential features are conserved [3]. In particular both proteins possess: i) multiple N-glycosylated sites; ii) an amino-terminal signal sequence that is removed in the mature protein; iii) a carboxy-terminal signal that is eliminated when the mature protein is linked to GPI, and iv) an N-terminal domain featured by tandem amino acid repeats (PHNPGY in avians, PHGGGWGQ in mammals) followed by a highly conserved hydrophobic core [3,4]. Differently from mammalian PrP, the digestion of ChPrP with trypsine or proteinase K produces peptide fragments stable to further proteolysis. One of these fragments comprises the 49129 sequence, consisting of a large part of the N-terminal domain [6]. This resistance to proteolysis may suggest a compact domain of the proline/glycine rich N-terminus, although the hexarepeat amino acid sequence seems to show no tendency towards a particular structured conformation [6]. More recently, NMR results showed that the ChPrP globular domain contains three α-helices and two short antiparallel β-sheets, with a spatial arrangement similar to that observed in mammalian PrP [7]. The N-terminal domain results to be flexible and unstructured and the 50-73 residues, encompassing part of the tandem hexarepeat sequence, have not yet been individually assigned by NMR [7]. Besides the wide conformational freedom of the N-terminal backbone, the significant content of proline residues may in principle bring about further complications in the structure determination of this part of protein, due to the presence of an equilibrium between cis and trans form in each XxxPro peptide bond, even if the stabilization of a preferred isomer may be assisted by other residue side chains, particularly histidine and tyrosine. In native and folded proteins the Xxx-Pro bond exists essentially in either cis or trans form because the interactions with neighbouring groups favours one of them, but an equilibrium exists in unfolded proteins. In small peptides containing proline residues, the two forms are almost