Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 117, Issue 11, Pages 5883-5894Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1912055117
Keywords
liquid-liquid phase separation; NMR spectroscopy; protein interactions; molecular simulation
Categories
Funding
- National Institute of General Medical Sciences (NIGMS) [R01GM118530]
- NSF [1845734, TG-MCB120014]
- ALS Association [17-IIP-342]
- NIGMS training grant [T32GM007601]
- US Department of Energy (DOE), Office of Science, Basic Energy Sciences, Division of Material Sciences and Engineering [DESC0013979]
- NIH [DP2GM105448, R35GM118082]
- Deutsche Forschungsgemeinschaft [SCHM 3082/2-1]
- Division of Biology and Medicine, Brown University
- Office of Science of the US Department of Energy [DE-AC02-05CH11231]
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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.
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