Structure and Dynamics of the Misfolding Intermediate in the Pathogenic T183A Prion Protein Mutant

Characterizing high-energy conformations in protein molecules is crucial to delineate the nature of dynamic processes that underlie biological activity, as these elusive species often play critical roles in fundamental mechanisms of the cell, such as enzyme catalysis or protein folding and aggregation, among many others. In this context, the integration of molecular simulations with experimental biophysics represents a powerful strategy to delineate complex conformational landscapes, enabling the study of transient conformations at atomic resolution. Here, we characterized intermediate states along the misfolding pathway of the human prion protein (PrP) variant T183A, which is associated with familial Creutzfeldt–Jakob disease. Using replica-averaged molecular dynamics simulations, restrained with nuclear magnetic resonance chemical shifts, we obtained structural ensembles showing enhanced conformational heterogeneity for the T183A variant compared with the WT protein. The mutant ensemble was found to populate partially misfolded states characterized by disruption of the β-sheet and local unfolding of key helical regions of the protein. Additionally, dynamic cross-correlation analyses evidenced significant loss of cooperative fluctuations across secondary structure elements, delineating how structural destabilization in the T183A variant leads to the insurgence of misfolding intermediates. Collectively, these findings provide critical insights into the underlying mechanisms of T183A-induced PrP misfolding and its consequent aggregation into amyloid fibrils.