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

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Liquid-liquid phase separation (LLPS) is crucial for membraneless organelle (MLO) formation in RNA processing.
• The RNA-binding protein TDP-43 is implicated in MLOs and amyotrophic lateral sclerosis (ALS) pathogenesis.
• ALS-associated TDP-43 mutations can disrupt its self-interaction and function.

Purpose of the Study:

• To investigate how designed single mutations in TDP-43 can modulate its helical structure to enhance assembly and function.
• To explore the role of specific glycine residues in inhibiting TDP-43 helical extension and helix-helix interactions.
• To understand the impact of TDP-43 mutations on LLPS, MLO fluidity, and splicing function.

Main Methods:

• Molecular simulation and NMR spectroscopy to analyze TDP-43 structure and dimerization.
• In vitro phase separation assays to assess TDP-43 assembly.
• Cellular assays using reporter compartments to measure fluidity of phase-separated TDP-43.
• Minigene assays to evaluate TDP-43 splicing function.

Main Results:

• Two conserved glycine residues (G335 and G338) inhibit TDP-43 helical extension and interactions.
• Variants at G335/G338, including ALS-associated G335D, partially relieve this inhibition.
• Substitution to alanine (G335A) significantly enhances TDP-43 in vitro phase separation.
• G335A mutation decreases the fluidity of phase-separated TDP-43 compartments in cells.
• G335A mutation enhances TDP-43 splicing function in a minigene assay.

Conclusions:

• The helical region of TDP-43 is a tunable module for controlling protein assembly and function.
• Biophysical principles can be applied to precisely modulate TDP-43 behavior in LLPS.
• Findings provide insights into ALS pathogenesis and potential applications in synthetic biology.