Phosphatidic Acid Vesicles Convert the Prion Protein into β-Sheet Fibrils with a Thermostable Structure but Temperature-Sensitive Packing

Abstract

Biomolecular cofactors, including nucleic acids and lipids, promote conversion of the cellular prion protein (PrPC) into pathogenic assemblies (prion scrapie, PrPSc), yet the mechanistic role of specific membrane lipids in the earliest stages remains unclear. Previous work showed that phosphatidic acid (PA) vesicles interact in vitro with murine recombinant PrP (recPrP) and trigger PrP aggregation without additional cofactors. Here, we dissect the PA-driven conversion pathway by monitoring PrP assembly and aggregate stability using spectroscopy and by comparing full-length recPrP with an N-terminally truncated construct. PA vesicles induced aggregation in both PrP constructs with similar efficiency, indicating that PA-mediated conversion does not require residues 23–89. Thermal stability analyses revealed a thermally resilient β-structured state accompanied by temperature-sensitive, partially reversible ThT binding, which we interpret as remodelling of higher-order packing. Time-resolved measurements showed that assembly reached a plateau within the measurement time resolution at all temperatures tested, with no resolvable lag phase. At plateau, the extent of ThT-positive assembly was greatest under conditions of higher bilayer order, while light scattering and secondary-structure content were comparable across temperatures. PrP alone showed no detectable temperature-dependent secondary-structure changes between 10 and 40 °C, consistent with membrane organization contributing to assembly architecture. Together, these results establish PA vesicles as a tractable model for interrogating cofactor-driven early prion aggregation and support membrane lipids and their physical state as modulators of PrP conversion into β-structured assemblies, with implications for mechanistic studies and screening strategies targeting lipid–protein interactions.