ALS-associated mutations reveal a conserved C-terminal self-assembly module in the TDP-43 low-complexity domain

TAR DNA-binding protein 43 (TDP-43) is an RNA-binding protein whose low-complexity domain (LCD) mediates self-assembly, liquid-liquid phase separation, and pathological aggregation associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Previous studies have established the central ultraconserved region (UCR) of the LCD as a major determinant of TDP-43 self-association. However, the contribution of the evolutionarily conserved C-terminal region remains poorly understood. To identify LCD residues required for self-assembly, we systematically analyzed 47 ALS-associated substitutions distributed throughout the TDP-43 LCD using hydrogel-binding and thioflavin T (ThT) assays. Several ALS-associated substitutions within the C-terminal conserved region (CCR) markedly reduced hydrogel incorporation and impaired fibrillization. Similar assembly defects were observed for methionine-to-serine substitutions at two conserved CCR residues, M405 and M414. Furthermore, CCR-containing C-terminal fragments encompassing residues 360-414 or 379-414 formed ThT-positive fibrillar assemblies in vitro . The assembly kinetics exhibited a characteristic lag phase followed by rapid fluorescence growth, consistent with the formation of cross-β-rich assemblies. These findings identify the CCR as an additional conserved self-assembly module within the TDP-43 LCD and expand the prevailing UCR-centered model of TDP-43 assembly. The observation that multiple ALS-associated substitutions reduce rather than enhance self-association further raises the possibility that disruption of normal TDP-43 assembly contributes to disease pathogenesis.