TAR DNA-binding protein 43 (TDP-43) liquid-liquid phase separation is mediated by just a few aromatic residues - PubMed (original) (raw)
TAR DNA-binding protein 43 (TDP-43) liquid-liquid phase separation is mediated by just a few aromatic residues
Hao-Ru Li et al. J Biol Chem. 2018.
Abstract
Eukaryotic cells contain distinct organelles, but not all of these compartments are enclosed by membranes. Some intrinsically disordered proteins mediate membraneless organelle formation through liquid-liquid phase separation (LLPS). LLPS facilitates many biological functions such as regulating RNA stability and ribonucleoprotein assembly, and disruption of LLPS pathways has been implicated in several diseases. Proteins exhibiting LLPS typically have low sequence complexity and specific repeat motifs. These motifs promote multivalent connections with other molecules and the formation of higher-order oligomers, and their removal usually prevents LLPS. The intrinsically disordered C-terminal domain of TAR DNA-binding protein 43 (TDP-43), a protein involved in motor neuron disease and dementia lacks a dominant LLPS motif, however, and how this domain forms condensates is unclear. Using extensive mutagenesis of TDP-43, we demonstrate here that three tryptophan residues and, to a lesser extent, four other aromatic residues are most important for TDP-43 to undergo LLPS. Our results also suggested that only a few residues may be required for TDP-43 LLPS because the α-helical segment (spanning ∼20 residues) in the middle part of the C-terminal domain tends to self-assemble, reducing the number of motifs required for forming a multivalent connection. Our results indicating that a self-associating α-helical element with a few key residues regulates condensate formation highlight a different type of LLPS involving intrinsically disordered regions. The C-terminal domain of TDP-43 contains ∼50 disease-related mutations, with no clear physicochemical link between them. We propose that they may disrupt LLPS indirectly by interfering with the key residues identified here.
Keywords: TAR DNA-binding protein 43 (TDP-43) (TARDBP); amyotrophic lateral sclerosis (ALS) (Lou Gehrig disease); intrinsically disordered protein; liquid-liquid phase separation; nuclear magnetic resonance (NMR); protein folding; protein self-assembly.
© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
Conflict of interest statement
The authors declare that they have no conflicts of interest with the contents of this article
Figures
Figure 1.
Amino acid sequence of the C-terminal domain of TDP-43 and the prediction of structural disorder and sequence complexity. A, the (G/S)-(F/Y)-(G/S) motif is colored in orange and the three tryptophans are colored purple. Positive and negatively charged residues are shown in blue and red, respectively. The locations of known ALS-associated mutations are indicated with stars. Those close to the motifs are shown in enlarged font. B, disorder predictions using the PONDR VSL2 (green) and VL3 (red) (62, 63), and the IUPRED (orange) (64) algorithms based on the primary sequence. Sequence complexity was calculated using the SEG algorithm (65) and the two main regions predicted with low sequence complexity are indicated with blue and purple bars, respectively.
Figure 2.
Liquid–liquid phase separation of the C-terminal domain of TDP-43. A, the optical density at 600 nm (OD600 nm) of a 20 μ
m
WT sample at different temperatures, highlighting the reversibility of the process. B, micrographs (scale bar: 10 μm) at different static temperatures. C, time-lapse micrographs demonstrating the reversibility of condensate formation (see “Experimental procedures” for details of how these images were collected). D, turbidity measured at 5 °C for different mutants in a 10 m
m
phosphate buffer at pH 6.5 only (solid bars) or with additional compounds (NaCl, urea, or hexanediol, open bars). E, micrographs from the samples whose turbidity is shown in panel D.
Figure 3.
Turbidity of samples of WT TDP-43 and tryptophan-to-glycine variants. All data were collected at 5 °C. A and B, turbidity of 20 μ
m
protein samples (A) in the absence and (B) in the presence of 100 m
m
NaCl. C, turbidity of 100 μ
m
protein samples in the absence of 100 m
m
NaCl. The gray bars indicate samples that precipitated before measurements could be taken, the error bars represent the standard deviation of three repeated measurements (blue squares). D, micrographs of the variants for which there is clear evidence of condensation in panels B and C (scale bar, 10 μm). E, schematic representations of the constructs. F, comparison of HSQC spectra between W334G and Δ3W. The HSQC spectrum of the W334G variant (orange) is overlaid on the Δ3W mutant (purple). Most of the cross-peaks overlap apart from those close to the mutation sites. The cross-peaks from residues in the α-helical region are highlighted.
Figure 4.
Turbidity of samples of WT TDP-43 and W334A and other phenylalanine-to-glycine and tyrosine-to-glycine variants. All data were collected at 5 °C. A and B, turbidity of 20 μ
m
protein samples (A) in the absence and (B) in the presence of 100 m
m
NaCl. C, turbidity of 100 μ
m
protein samples in the absence of 100 m
m
NaCl. The gray bars indicate samples that precipitated before measurements could be taken, the error bars represent the standard deviation of three repeated measurements (blue squares). D, comparison of micrographs of some of the variants indicated with gray stars in panel C (scale bar, 10 μm). E, schematic representations of the constructs.
Figure 5.
NMR signal intensity ratios and chemical shift perturbations between 100 and 20 μm protein samples. A, signal intensity ratios and B, chemical shift perturbations between 100 and 20 μ
m
samples of W334G, W334A, and Δ2W.Δ3F.Δ1Y variants of TDP-43. C, HSQC spectra of the 20 μ
m
(red) and 100 μ
m
(green) samples of the Δ2W.Δ3F.Δ1Y variant. The most pronounced changes in chemical shift are highlighted.
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