Phosphomimetic substitutions in TDP-43's transiently α-helical region suppress phase separation

Biophys J. 2024 Jan 4:S0006-3495(24)00001-8. doi: 10.1016/j.bpj.2024.01.001. Online ahead of print.ABSTRACTPhosphorylated TAR DNA-binding protein of 43 kDa (TDP-43) is present within the aggregates of several age-related neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD) and Alzheimer's disease, to the point that the presence of phosphorylated TDP-43 is considered a hallmark of some of these diseases. The majority of known TDP-43 phosphorylation sites detected in ALS/FTLD patients is located in the low complexity domain (LCD), the same domain that has been shown to be critical for TDP-43 liquid-liquid phase separation (LLPS). However, the effect of these LCD phosphorylation sites on TDP-43 LLPS has been largely unexplored, and any work that has been done has largely focused on sites near the C-terminal end of the LCD. Here, we used a phosphomimetic approach to explore the impact of phosphorylation at residues S332 and S333, sites located within the transiently α-helical region of TDP-43 that have been observed to be phosphorylated in disease, on protein LLPS. Our turbidimetry and fluorescence microscopy data demonstrate that these phosphomimetic substitutions greatly suppress LLPS, and solution NMR data strongly suggest that this effect is at least in part due to the loss of α-helical propensity of the phosphomimetic protein variant. We also show that the S332D and S333D substitutions slow TDP-43 LCD droplet ag...
Source: Biophysical Journal - Category: Physics Authors: Source Type: research