Probing the structure of the functional helical assembly of the ALS-associated protein TDP-43

TDP-43 is an essential nuclear protein involved in RNA metabolism, and its aggregation is implicated in many neurodegenerative diseases like amyotrophic lateral sclerosis and frontotemporal lobar dementia. Many functions of TDP-43 are performed in dynamic cellular granules formed by liquid-liquid phase separation (LLPS). About 90% ALS associated mutations are located at in the aggregation-prone C-terminal domain (CTD), highlighting its importance in TDP-43 dysfunction. We previously showed that a conserved region (residues 319-342) of the aggregation-prone TDP-43 CTD forms alpha-helical structure that contributes significantly to LLPS and is important for function. Although phase separation of TDP-43 has been linked to its function and its disruption of aggregation and disease, the residue-level determinants of TDP-43 CTD phase separation remain elusive. Here we show that covalently linked CTD dimers oligomerize into higher-order helical assemblies. Based on comparing cross-linking sites, we conclude that the structure contains parallel helix structures in the conserved region. Analytical ultracentrifugation (AUC) showed the CTD helical assembly consists of tetrameric and octameric assemblies. NMR NOE experiments demonstrate that significant contacts are formed between alanine, methionine, leucine, and tryptophan residues, enabling us to build structural models of the assembly.