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

Proceedings of the National Academy of Sciences of the United States of America
Alexander E ConicellaNicolas L Fawzi

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 assemb...Continue Reading

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Methods Mentioned

BETA
NMR
fluorescence recovery after photobleaching
Fluorescence
PCR
size-exclusion chromatography
protein folding

Key Resources (RRID) Mentioned

CVCL_0063

Software Mentioned

- Sparky
Mathematica
SPARTA +
Blue
ImageJ
HOOMD
NMRFAM
NMRPipe
GraphPad Prism
Leica Application Suite X

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