18Prions are pathological isoforms of the cellular prion protein (PrP C ) responsible for transmissible 19 spongiform encephalopathies (TSE). PrP C interacts with copper through unique octarepeat and 20 non-octarepeat (non-OR) binding sites. Previous works on human PrP C suggest that copper 21 binding to the non-OR region may have a role during prion conversion. The molecular details of 22 copper coordination within the non-OR region are not well characterized. By means of small 23 angle X-ray scattering (SAXS) and extended X-ray absorption fine structure (EXAFS) 24 spectroscopy, we have investigated the Cu(II) structural effects on the protein folding and its 25 coordination geometries when bound to the non-OR region of recombinant PrP C (recPrP) from 26 animal species considered high or less resistant to TSE. As TSE-resistant model, we used ovine 27 PrP C carrying the protective polymorphism at residues A136, R154 and R171 (OvPrP ARR); 28 while as highly TSE-susceptible PrP C models we employed OvPrP with polymorphism V136,
29R154 and Q171 (OvPrP VRQ) and Bank vole recPrP (BvPrP). Our results reveal that Cu(II) 30 affects the structural plasticity of the non-OR region leading to a more compacted conformation 31 of recPrP. We also identified two Cu(II) coordinations in the non-OR region of these animal 32 species. In type-1 coordination present in OvPrP ARR, Cu(II) is coordinated by four residues 33 (S95, Q98, M109 and H111). Conversely, the type-2 coordination is present in OvPrP VRQ and 34 BvPrP, where Cu(II) is coordinated by three residues (Q98, M109 and H111) and by one water 35 molecule, making the non-OR region more flexible and open to the solvent. These changes in 36 copper coordination in prion resistant and susceptible species provide new insights into the 37 molecular mechanisms governing the resistance or susceptibility of certain species to TSE. Keywords 42 Prion protein, SAXS, Ensemble Optimization Method, EXAFS spectroscopy, non-OR region, 43 copper coordination, transmissible spongiform encephalopathies.44 45 48 of the physiological cellular prion protein (PrP C ) into a β-sheet rich isoform called prion or PrP Sc 49(1). TSE are rare disorders that can be sporadic, genetic or infectious. Animal TSE include 50 scrapie in sheep and goats, chronic wasting disease (CWD) in cervids and bovine spongiform 51 encephalopathy (BSE) in cattle (2).
52The PrP C structure consists in a C-terminal folded domain (from residues 128 to 231, hereafter in 53 human numbering) mainly containing α-helical motifs and two short anti-parallel β-sheets (3).
54On the contrary, the N-terminal moiety (residues 23-127) is largely unstructured (4) and features 55 an octapeptide-repeat region (OR) (residues 61-91) composed by four octapeptides, each 56 carrying histidines able to coordinate prevalently one copper ion, Cu(II) (5). Cu(II) can also bind 57 at two additional histidines -H96 and H111 with coordination from the imidazole rings and 58 nearby backbone amides-located in a segment (residues 90-111) called "fifth" or n...