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

Journal Article · Tue Mar 03 23:00:00 EST 2020 · Proceedings of the National Academy of Sciences of the United States of America

DOI:https://doi.org/10.1073/pnas.1912055117· OSTI ID:1603181

Conicella, Alexander E. ^[1]; Dignon, Gregory L. ^[2]; Zerze, Gül H. ^[3]; Schmidt, Hermann Broder ^[4]; D’Ordine, Alexandra M. ^[5]; Kim, Young C. ^[6]; Rohatgi, Rajat ^[7]; Ayala, Yuna M. ^[8]; Mittal, Jeetain ^[9]; Fawzi, Nicolas L. ^[10]

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1. Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, RI 02912,, Graduate Program in Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI 02912,, Department of Chemistry, University of Toronto, Toronto, ON M5S 1A8, Canada,
2. Department of Chemical and Biomolecular Engineering, Lehigh University, Bethlehem, PA 18015,, Laufer Center for Physical and Quantitative Biology, Stony Brook University, Stony Brook, NY 11794,
3. Department of Chemical and Biomolecular Engineering, Lehigh University, Bethlehem, PA 18015,, Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ 08540,
4. Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305,
5. Graduate Program in Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI 02912,
6. Center for Materials Physics and Technology, Naval Research Laboratory, Washington, DC 20375,
7. Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305,, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305,
8. Edward Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO 63103
9. Department of Chemical and Biomolecular Engineering, Lehigh University, Bethlehem, PA 18015,
10. Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, RI 02912,

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.

• Sponsoring Organization: USDOE
• Grant/Contract Number: SC0013979
• OSTI ID: 1603181
• Alternate ID(s): OSTI ID: 1625050
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Issue: 11 Vol. 117; ISSN 0027-8424
• Publisher: Proceedings of the National Academy of Sciences
• Country of Publication: United States
• Language: English