Hydrophobic residues in disordered and helical regions mediate the oligomerization and phase separation of TDP-43 C-terminal domain.

Abstract

TAR DNA binding protein 43 (TDP-43) is a nuclear, multidomain protein implicated in RNA metabolism. The cytoplasmic accumulation of TDP-43 can result in the formation of inclusion bodies which are a hallmark of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The prion-like, disordered C-terminal domain (CTD) of TDP-43 (spanning residues 267-414) is aggregation-prone and harbors the majority (∼90%) of all ALS-related mutations. Several studies report that CTD can undergo liquid-liquid phase separation (LLPS). The conserved, hydrophobic region (CR, spanning residues 319-341) in CTD populates an α-helical structure which is essential for phase separation. The requirement of CR for phase separation is attributed to its tendency to undergo helix-helix self-association and promote oligomerization. In addition to CR, aromatic residues in the flanking disordered regions (IDR1/2) also play a critical role in CTD self-assembly. However, the relative contributions of critical residue types in CR and flanking regions on the overall phase separation propensity of CTD are unclear. Here, we utilized multiscale simulations coupled with biophysical experiments to determine the role of hydrophobic residues in the CR and IDR1/2 regions on CTD phase separation. Single chain and condensed phase simulations collectively identified the contribution of aromatic residues while also suggesting significant contributions to LLPS by other residue types, which were previously not recognized as essential for phase separation within and outside the CR region. The computational observations were successfully corroborated by in vitro microscopy and saturation concentration measurements. Finally, NMR experiments determined that CTD oligomerization was critically dependent on these residues within CR and IDR1/2 regions. Overall, our work uncovers the collective importance of hydrophobic residues across both helical and disordered regions in modulating the oligomerization and phase separation of CTD.