Investigating the effect of progressive truncations at the ALS-linked protein TDP-43 RRM2 on its aggregation mechanism

Amyotrophic lateral sclerosis is a neurodegenerative disease characterized by inclusions of TDP-43 protein. C-terminal fragments (CTFs) of TDP-43, generated by cleavage within its second RNA recognition motif (RRM2), have been found forming aggregates in patients. Aggregation has often been attributed to the C-terminal domain, but increasing evidence indicates that RRM2 fragments contribute to pathological inclusions. We performed extensive molecular dynamics simulations to investigate the changes resulting from the truncation that could lead to aggregation. We analyzed the full RRM2 domain (fRRM2, residues 192–261) and two fragments commonly observed in CTFs (tRRM2A, residues 220–261, and tRRM2B, residues 209–261). We found that truncation results in distinct aggregation-prone states. tRRM2B appears to rely on (Formula presented)-sheet elements associated with amyloid-like aggregation, whereas tRRM2A exhibits higher structural variability and a reduced (Formula presented)-content, suggesting a phase separation-like aggregation mechanism. We further simulated an extended fragment of tRRM2A, tRRM2A-l (residues 220–269). Although its predicted aggregation propensity remains largely unchanged, tRRM2A-l exhibits increased structural flexibility, and a stronger exposure of Nuclear Export Signal residues. Our results indicate that subtle differences in RRM2 fragment length influence potential misfolding pathways. Future studies and therapeutic strategies to prevent TDP-43 aggregation should carefully consider the specific domain adopted.