TDP-43 α-helical structure tunes liquid–liquid phase separation and function

Significance

TDP-43 is an essential RNA-binding protein that assembles into protein inclusions in >95% of cases of amyotrophic lateral sclerosis (ALS). A partially helical region in the predominantly disordered C-terminal domain harbors several mutations associated with ALS and is important for TDP-43 function and liquid–liquid phase separation. We directly demonstrate that this helical region undergoes large structural changes upon helix–helix dimerization and that point mutations can enhance helix–helix assembly. Furthermore, we demonstrate that these point variants can be used to control the material properties of phase-separated TDP-43 constructs in cells and can enhance TDP-43 RNA-splicing function. Therefore, engineered forms of the TDP-43 helical domain could be used to control in-cell phase separation, dynamic assembly, and function.

Abstract

Liquid–liquid phase separation (LLPS) is involved in the formation of membraneless organelles (MLOs) associated with RNA processing. The RNA-binding protein TDP-43 is present in several MLOs, undergoes LLPS, and has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS). While some ALS-associated mutations in TDP-43 disrupt self-interaction and function, here we show that designed single mutations can enhance TDP-43 assembly and function via modulating helical structure. Using molecular simulation and NMR spectroscopy, we observe large structural changes upon dimerization of TDP-43. Two conserved glycine residues (G335 and G338) are potent inhibitors of helical extension and helix–helix interaction, which are removed in part by variants at these positions, including the ALS-associated G335D. Substitution to helix-enhancing alanine at either of these positions dramatically enhances phase separation in vitro and decreases fluidity of phase-separated TDP-43 reporter compartments in cells. Furthermore, G335A increases TDP-43 splicing function in a minigene assay. Therefore, the TDP-43 helical region serves as a short but uniquely tunable module where application of biophysical principles can precisely control assembly and function in cellular and synthetic biology applications of LLPS.