Pathological C‐terminal phosphomimetic substitutions alter the mechanism of liquid–liquid phase separation of TDP‐43 low complexity domain

C-terminally phosphorylated TAR DNA-binding protein of 43 kDa (TDP-43) marks the proteinaceous inclusions that characterize a number of age-related neurodegenerative diseases, including amyotrophic lateral sclerosis, frontotemporal lobar degeneration and Alzheimer's disease. TDP-43 phosphorylation at S403/S404 and (especially) at S409/S410 is, in fact, accepted as a biomarker of proteinopathy. These residues are located within the low complexity domain (LCD), which also drives the protein's liquid-liquid phase separation (LLPS). The impact of phosphorylation at these LCD sites on phase separation of the protein is a topic of great interest, as these post-translational modifications and LLPS are both implicated in proteinopathies. Here, we employed a combination of experimental and simulation-based approaches to explore this question on a phosphomimetic model of the TDP-43 LCD. Our turbidity and fluorescence microscopy data show that phosphomimetic Ser-to-Asp substitutions at residues S403, S404, S409 and S410 alter the LLPS behavior of TDP-43 LCD. In particular, unlike the LLPS of unmodified protein, LLPS of the phosphomimetic variants displays a biphasic dependence on salt concentration. Through coarse-grained modeling, we find that this biphasic salt dependence is derived from an altered mechanism of phase separation, in which LLPS-driving short-range intermolecular hydrophobic interactions are modulated by long-range attractive electrostatic interactions. Overall, this in vitro and in silico study provides a physiochemical foundation for understanding the impact of pathologically relevant C-terminal phosphorylation on the LLPS of TDP-43 in a more complex cellular environment.