Methionine oxidation alters both helical assembly and disordered contacts in human TDP-43 C-terminal domain phase separation

TAR DNA binding protein 43 (TDP-43), a key protein linked to ALS pathology, undergoes phase separation and forms functional assemblies via condensation within cells. The conserved region (CR) within its C-terminal domain (CTD) mediates self-assembly through helix-helix interactions, while the flanking intrinsically disordered regions (IDRs) contribute to phase separation through transient interactions involving aromatic and hydrophobic residues. The CTD contains ten methionine residues distributed equally between these regions, making it particularly susceptible to oxidative modifications. While methionine oxidation is known to impair phase separation, neither the precise mechanism nor the specific contribution of methionines in the CR compared to the IDRs has been determined. Here, we combine NMR spectroscopy and all-atom molecular dynamics (MD) simulations to reveal if and how methionine oxidation in each region differentially affects CTD structure and phase separation. We demonstrate that all methionine residues are vulnerable to oxidation, leading to distinct regional effects: oxidation of CR methionines disrupts helical structure and directly impairs intermolecular helical association, while oxidation of IDR methionines disrupts long-range contacts. Hence, oxidation of methionines in both regions contributes to impaired phase separation, albeit through different mechanisms. These findings establish methionines as critical redox-sensitive modulators of TDP-43 phase behavior and provide molecular insights into how oxidative stress may contribute to TDP-43 dysregulation in neurodegenerative diseases.