(467g) Revealing the Atomic-Level Details of Helix-Mediated Tdp-43 Oligomerization and Phase Separation

TAR DNA binding protein-43 (TDP-43) is a key nuclear RNA-binding protein implicated in RNA metabolism. The C-terminal domain (CTD) of TDP-43, which is intrinsically disordered and aggregation-prone, commonly found in cytoplasmic inclusions associated with neurodegenerative disorders including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). CTD undergoes phase separation in isolation, which is proposed to play essential roles in its cellular function such as splicing and nuclear retention. CTD contains a conserved helical region (CR, spanning residues 319-341) which adopts a transient α-helix structure and forms helix-helix contacts that drives its higher-order oligomerization and phase separation. Notably, ALS-associated mutations in the CR disrupt both oligomerization and phase separation, further highlighting the importance of the CTD in disease. Therefore, uncovering the atomistic details underlying the formation of CTD oligomerization and phase separation is crucial for the designing of novel therapeutic interventions for TDP-43 associated ALS/FTD. However, the high aggregation propensity and dynamic assembly of TDP-43 CTD hinders a high-resolution structural characterization of helix-helix contacts in CTD assembled states. In this study, we employ AlphaFold2-Multimer modeling and all-atom molecular dynamics (AAMD) simulations to obtain atomic structural insights into oligomeric and phase-separated assemblies of TDP-43 CTD. We have performed extensive AAMD simulations (aggregate time ~100 μs) of a TDP-43 fragment (aa: 310-350) containing the CR to characterize helix-mediated interactions of CTD dimers. We observed a good correlation of per-residue intermolecular contacts in the dimer state with the experimental saturation concentration (csat) for CTD phase separation obtained from position-by-position alanine scan of the CR residues. This demonstrates that helix-helix contacts in the dimer state are a major contributor to CTD phase separation and serve as the basis for the formation of dynamic, higher-order TDP-43 CTD assemblies. Through integrative modeling, we determined the first structural model of TDP-43 CR tetramer, which exhibits unique stability and shows a good agreement with NMR-derived structural measurements. Our findings offer mechanistic insights into TDP-43 functional assembly and provide a structural model for developing therapeutic interventions.