The anomalous phase separation of TAR DNA-binding protein 43 kDa (TDP-43), can lead to the formation of protein aggregates that are linked to neurodegenerative diseases, such as Alzheimer’s disease and amyotrophic lateral sclerosis (ALS). Therefore, unveiling the aggregation mechanism of these proteins is critical to understanding their pathological roles. Toward this goal, we scrutinized the aggregation behaviors of rational mutants of TDP-43 in cells using distributed amphifluoric FRET (DAmFRET), and revealed that a single proline at residue 363 (P363) strongly impacts the amyloid nucleation by TDP-43. Explicitly, any mutation to P363 immensely enhanced the aggregation. Hence, our hypothesis is that proline’s unique ability to populate a cis isoform prevents the aggregation of TDP, while this isoform does so in a dominant fashion as the great majority (∼90%) of molecules contain the highly amyloidogenic trans isoform. We further showed that the amyloid kinetics of synthetic TDP-43 (cis-P363) and TDP-43 (trans-P363) differ in vitro. Nevertheless, these experimental observations and our hypothesis demand a computational approach to unravel the mechanism behind TDP-43 aggregation. According to our unbiased, coarse grained and then all-atom simulations, the 100% trans P363 TDP protofibril (PDB id: 7PY2) derivative (273–414) is stable throughout the 1 us simulation, while in the cis protofibril, a capping phenomenon is observed. Due to capping at the end, in contrast to trans, a cis protofibril is unable to grow further. In other words, cis prevents TDP aggregation. Moreover, MD simulations reveal that this capping ability of cis proline is highly correlated with the P363(CA)-P363(N)-E362(C)-E362(CA) dihedral present in TDP-43, that determines trans(or cis) isoforms. Eventually our MD simulation techniques combined with analysis will be used to unravel the mechanism by which cis/trans isomerization of P363 governs/attenuates pathologic TDP-43 aggregation.
Hettige et al. (2026) studied this question.