Structural details of helix-mediated TDP-43 C-terminal domain multimerization.

The primarily disordered C-terminal domain (CTD) of TAR DNA binding protein-43 (TDP-43), a key nuclear protein in RNA metabolism, forms neuronal inclusions in several neurodegenerative diseases. A conserved region (CR, spanning residues 319-341) in CTD forms transient helix-helix contacts important for its higher-order oligomerization and function that are disrupted by ALS-associated mutations. However, the structural details of CR assembly and the explanation for several ALS-associated variants' impact on phase separation and function remain unclear due to challenges in analyzing the dynamic association of TDP-43 CTD using traditional structural biology approaches. By employing an integrative approach, combining biophysical experiments, biochemical assays, AlphaFold2-Multimer (AF2-Multimer), and atomistic simulations, we generated structural models of helical oligomerization of TDP-43 CR. Using NMR, we first established that the native state of TDP-43 CR under physiological conditions is α-helical. Next, alanine scanning mutagenesis revealed that while hydrophobic residues in the CR are important for CR assembly, phase separation and TDP-43 nuclear retention function, polar residues down regulate these processes. Finally, pairing AF2-Multimer modeling with AAMD simulations indicated that dynamic, oligomeric assemblies of TDP-43 that are stabilized by a methionine-rich core with specific contributions from a tryptophan/leucine pair. In conclusion, our results advance the structural understanding of the mechanisms driving TDP-43 function and provide a window into the initial stages of its conversion into pathogenic aggregates.