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.
Conicella, A. E., Dignon, G. L., Zerze, G. H., Schmidt, H. B., D'Ordine, A. M., Kim, Y. C., ... Fawzi, N. L.
citation count
134
complete list of authors
Conicella, Alexander E||Dignon, Gregory L||Zerze, Gül H||Schmidt, Hermann Broder||D'Ordine, Alexandra M||Kim, Young C||Rohatgi, Rajat||Ayala, Yuna M||Mittal, Jeetain||Fawzi, Nicolas L
