Charge Neutralization of the Central Lysine Cluster in Prion Protein (PrP) Promotes PrPSc-like Folding of Recombinant PrP Amyloids

The structure of the infectious form of prion protein, PrPSc, remains unclear. Most pure recombinant prion protein (PrP) amyloids generated in vitro are not infectious and lack the extent of the protease-resistant core and solvent exclusion of infectious PrPSc, especially within residues similar to 90-160. Polyanionic cofactors can enhance infectivity and PrPSc-like characteristics of such fibrils, but the mechanism of this enhancement is unknown. In considering structural models of PrPSc multimers, we identified an obstacle to tight packing that might be overcome with polyanionic cofactors, namely, electrostatic repulsion between four closely spaced cationic lysines within a central lysine cluster of residues 101-110. For example, in our parallel in-register intermolecular beta-sheet model of PrPSc, not only would these lysines be clustered within the 101-110 region of the primary sequence, but they would have intermolecular spacings of only similar to 4.8 between stacke beta-strands. We have now performed molecular dynamics simulations predicting that neutralization of the charges on these lysine residues would allow more stable parallel in-register packing in this region. We also show empirically that substitution of these clustered lysine residues with alanines or asparagines results in recombinant PrP amyloid fibrils with extended proteinase-K resistant beta-sheet cores and infrared spectra that are more reminiscent of bona fide PrPSc. These findings indicate that charge neutralization at the central lysine cluster is critical for the folding and tight packing of N-proximal residues within PrP amyloid fibrils. This charge neutralization may be a key aspect of the mechanism by which anionic cofactors promote PrPSc formation.