ALS Mutations Disrupt Phase Separation Mediated by α-Helical Structure in the TDP-43 Low-Complexity C-Terminal Domain

Highlights

• Cooperative helix formed by TDP-43 residues 321–330

• Helix and conserved residues 331–340 essential for liquid-liquid phase separation

• ALS mutations within 321–340 alter phase separation and disrupt self-interaction

• Helical structure enhanced by self-interaction of the 321–340 region

Summary

RNA-binding protein TDP-43 mediates essential RNA processing but forms cytoplasmic neuronal inclusions via its C-terminal domain (CTD) in amyotrophic lateral sclerosis (ALS). It remains unclear if aggregated TDP-43 is neurotoxic and if ∼50 ALS-associated missense mutations in TDP-43 CTD promote aggregation, or if loss of normal function plays a role in disease. Recent work points to the ability of related proteins to assemble into functional phase-separated ribonucleoprotein granules via their structurally disordered prion-like domains. Here, we provide atomic details on the structure and assembly of the low-complexity CTD of TDP-43 into liquid-liquid phase-separated in vitro granules and demonstrate that ALS-associated variants disrupt interactions within granules. Using nuclear magnetic resonance spectroscopy, simulation, and microscopy, we find that a subregion cooperatively but transiently folds into a helix that mediates TDP-43 phase separation. ALS-associated mutations disrupt phase separation by inhibiting interaction and helical stabilization. Therefore, ALS-associated mutations can disrupt TDP-43 interactions, affecting function beyond encouraging aggregation.