(169ap) Atomic-Level Structural Model of Helical Tdp-43 Oligomers

TAR DNA binding protein-43 (TDP-43) is a key nuclear RNA-binding protein implicated in RNA metabolism such as transcription and splicing. The C-terminal domain (CTD) of TDP-43 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 contains a conserved helical region (CR, spanning residues 319-341) which adopts a transient α-helix structure and drives the formation of higher-order oligomers which are important for its splicing and nuclear retention. Notably, ALS-associated mutations in the CR disrupt oligomerization and function, further highlighting the importance of the CTD in disease. Therefore, uncovering the atomistic details underlying the formation of helix-mediated CTD oligomers is crucial for the development of novel therapeutic strategies for ALS/FTD. However, achieving a high-resolution, structural characterization of these high-order oligomers remains challenging, mainly due to CTD’s high aggregation propensity and dynamic assembly. To overcome these challenges, we employed an integrative modeling strategy based on state-of-the-art computational methods including AlphaFold2-Multimer modeling and all-atom molecular dynamics (AAMD) simulations, along with experimental insights from biophysical experiments, to probe the atomic structural details of TDP-43 helical self-assembly. We first characterized the structural ensemble of the TDP-43 CR dimer state (aggregate time ~100 μs), demonstrating the essential role of helix-helix contacts in driving the formation of dynamic TDP-43 CTD higher-order assemblies. Our simulations suggest that CR dimers lack a stable orientation and instead exhibit dynamically interconverting structures with heterogeneous helix-helix interactions. Through integrative modeling, we identified a uniquely stable atomic structural model of the TDP-43 CR tetrameric state, in qualitative agreement with experimental measurements from NMR spectroscopy. Importantly, our structural model elucidates, for the first time, the crucial structural role of the G335-Q343 region, a hotspot of ALS mutations, which becomes partially helical only upon higher-order assembly. Overall, our study makes significant advances towards structural characterization of TDP-43 functional assemblies and provides clues regarding their transition into pathogenic aggregates.