Aliphatic residues contribute significantly to the phase separation of TDP-43 C-terminal domain

TAR DNA binding protein 43 (TDP-43) is involved in key processes in RNA metabolism such as splicing, stability and transcription. TDP-43 dysfunction is frequently implicated in many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD). The prion-like, disordered C-terminal domain (CTD) of TDP-43 is aggregation-prone and harbors the majority (~90%) of all ALS-related mutations. Recent studies have established that TDP-43 CTD can undergo liquid-liquid phase separation (LLPS) in isolation and is important for phase separation (PS) of the full-length protein under physiological conditions. While a short conserved helical region (CR, spanning residues 319-341) promotes oligomerization and is essential for LLPS, aromatic residues in the flanking disordered regions (IDR1/2) have also been found to play a critical role in PS and aggregation. However, TDP-43 CTD has a distinct sequence composition compared with other phase separating proteins, including many aliphatic residues. These residues have been suggested to modulate the apparent viscosity of the resulting phases, but their direct contribution to phase separation has been relatively ignored. Here, we utilized a multiscale simulation and experimental approach to assess the residue-level determinants of TDP-43 CTD phase separation. Single chain and condensed phase simulations performed at the atomistic and coarse-grained level respectively, identified the importance of aromatic residues (previously established) while also suggesting an essential role for aliphatic methionine residues in LLPS. In vitro experiments confirmed the role of phenylalanine, methionine, and leucine (but not alanine) residues in driving the phase separation of CTD, which have not been previously considered essential for describing the molecular grammar of PS. Finally, NMR experiments also showed that phenylalanine residues in the disordered flanking regions and methionine residues both within and outside the CR contribute important contacts to CTD interactions. Broadly, our work highlights the importance of non-alanine aliphatic residues such as methionine and leucine, and potentially valine and isoleucine, in determining the LLPS propensity, expanding the molecular grammar of protein phase separation to include critical contributions from aliphatic residues.