Investigating the interaction of TDP-43 with RNA using structural mass spectrometry

Deposits of TDP-43, a disordered RNA-binding protein, in the brain is hallmark of amyotrophic lateral sclerosis (ALS). Binding of TDP-43 to RNA protects from aggregation. However, the structure(s) and conformational dynamics of monomeric TDP-43, its higher order assemblies, and the TDP-43-RNA complex remain elusive, despite their importance in understanding disease progression. Here we have used a combination of biophysical analyses and powerful mass spectrometry (MS) techniques to interrogate the conformational dynamics of monomeric full-length TDP-43 and its assemblies with RNA. Our results from HDX-MS show that, in the presence of RNA we observe allosteric protection from exchange in areas associated with amyloid formation suggesting a possible structural rearrangement that protects RNA bound TDP-43 from aggregation. We also identify allosteric changes within the short alpha helix motif located in the CTD upon RNA binding. These allosterically altered regions are known to influence the ability of TDP-43 to self-assemble and can fine-tune its RNA binding repertoire. Taken together, these data reveal new insights into the structure and dynamics of TDP-43, alongside the intra-protein and protein-RNA interactions which likely play a key role in fine tuning TDP-43 self-assembly into both condensates and amyloid fibrils. Understanding the molecular mechanism of these processes is key to elucidating the role of TDP-43 in the pathology of ALS and FTLD and may lead to new therapeutics to treat these diseases.