Human prion proteins and mutants through the lens of systems biology

This is the authors' abstract. We don't add key points for this paper.

Autocatalytic conformational changes of cellular prion proteins (PrP C ) to the β-sheet-enriched scrapie isoform (PrP SC ) drive prion diseases, yet the precise molecular mechanisms remain unclear. To address this, we employ a multi-pronged framework integrating network-theoretic analyses, molecular dynamics (MD) simulations, and experimental validation to characterize critical residues governing human PrP (huPrP) structural integrity. Residue Interaction Networks (RINs) of: (A) wild-type (WT) huPrP, and, (B) five disease-associated point mutants (Met166Val, Glu220Lys, Glu200Lys, Asp178Asn, and Ser170Asn) were evaluated using three rigorous network methods: centrality measures, S hortest P ath A lteration F raction (SPAF), and D ifferential N etworks (DN). Across all the diverse point mutations, five residues (Met206, Val209, Val210, Met213, and Cys214) consistently exhibit high centrality scores. SPAF and DN analyses demonstrate that alterations at these positions disrupt connectivity and increase the propensity for misfolding and protease resistance. To validate these topological findings, key residues within the C-terminal domain (121-230) were substituted via site-directed mutagenesis. Thermal denaturation monitored by circular dichroism (CD) spectroscopy reveals that the Met213Ala substitution significantly lowers the melting temperature, T m , relative to the WT, whereas the Val210Ala variant unfolds with a T m comparable to the WT. Furthermore, MD simulations of de novo AlphaFold-predicted mutant structures corroborate these findings. Mutants exhibit greater structural flexibility and deviation, with average root Mean Square Deviations rising from 0.18 nm (WT) to 0.32 nm (Cys214Ala), 0.29 nm (Met206Ala), and 0.25 nm (Val210Lys). This integration of theoretical and experimental techniques provides a robust framework for identifying critical determinants of protein stability.