ALS-Causing Mutations Significantly Perturb the Self-Assembly and Interaction with Nucleic Acid of the Intrinsically Disordered Prion-Like Domain of TDP-43 - PubMed (original) (raw)

ALS-Causing Mutations Significantly Perturb the Self-Assembly and Interaction with Nucleic Acid of the Intrinsically Disordered Prion-Like Domain of TDP-43

Liangzhong Lim et al. PLoS Biol. 2016.

Abstract

TAR-DNA-binding protein-43 (TDP-43) C-terminus encodes a prion-like domain widely presented in RNA-binding proteins, which functions to form dynamic oligomers and also, amazingly, hosts most amyotrophic lateral sclerosis (ALS)-causing mutations. Here, as facilitated by our previous discovery, by circular dichroism (CD), fluorescence and nuclear magnetic resonance (NMR) spectroscopy, we have successfully determined conformations, dynamics, and self-associations of the full-length prion-like domains of the wild type and three ALS-causing mutants (A315E, Q331K, and M337V) in both aqueous solutions and membrane environments. The study decodes the following: (1) The TDP-43 prion-like domain is intrinsically disordered only with some nascent secondary structures in aqueous solutions, but owns the capacity to assemble into dynamic oligomers rich in β-sheet structures. By contrast, despite having highly similar conformations, three mutants gained the ability to form amyloid oligomers. The wild type and three mutants all formed amyloid fibrils after incubation as imaged by electron microscopy. (2) The interaction with nucleic acid enhances the self-assembly for the wild type but triggers quick aggregation for three mutants. (3) A membrane-interacting subdomain has been identified over residues Met311-Gln343 indispensable for TDP-43 neurotoxicity, which transforms into a well-folded Ω-loop-helix structure in membrane environments. Furthermore, despite having very similar membrane-embedded conformations, three mutants will undergo further self-association in the membrane environment. Our study implies that the TDP-43 prion-like domain appears to have an energy landscape, which allows the assembly of the wild-type sequence into dynamic oligomers only under very limited condition sets, and ALS-causing point mutations are sufficient to remodel it to more favor the amyloid formation or irreversible aggregation, thus supporting the emerging view that the pathologic aggregation may occur via the exaggeration of functionally important assemblies. Furthermore, the coupled capacity of TDP-43 in aggregation and membrane interaction may critically account for its high neurotoxicity, and therefore its decoupling may represent a promising therapeutic strategy to treat TDP-43 causing neurodegenerative diseases.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1

Fig 1. Characterization of the wild-type TDP-43 prion-like domain.

(A) Domain organization of the 414-residue TDP-43 protein, which is composed of the N-ubiquitin-like domain, nuclear localization signal (L), two RNA recognition motifs (RRM1 and RRM2) hosting a nuclear export signal (E), and C-terminal prion-like domain abundant in Gln/Asn/Ser/Gly residues. (B) Far-UV CD spectra collected at 25°C for the prion-like domain over Lys263-Met414 at protein concentrations of 20 μM in Milli-Q water at pH 4.0 and in 1 mM phosphate buffer at pH 6.8 at different time points. (C) Superimposition of the two-dimensional NMR 1H-15N HSQC spectra of the prion-like domain acquired at 25°C, and at a protein concentration of 100 μM in Milli-Q water at pH 4.0 (blue), and in 1 mM phosphate buffer at pH 5.0 after 15 min (red). Green box is used to indicate the HSQC peaks of Gly residues while green arrows are used to indicate the HSQC peaks disappeared at pH 5.0. (D) Superimposition of HSQC spectra of the prion-like domain at 25°C in Milli-Q water at pH 4.0 (blue) and in 1 mM phosphate buffer at pH 6.8 after 15 min (red). Pink arrows are used to indicate the HSQC peaks manifested at pH 6.8. (E) Superimposition of HSQC spectra of the prion-like domain at 25°C in 1 mM phosphate buffer at pH 6.8 after 15 min (blue) and after 4 hr (red). (F) Superimposition of HSQC spectra of the prion-like domain at 25°C in 1 mM phosphate buffer at pH 6.8 after 15 min (blue) and after 9 hr (red). (G) Superimposition of HSQC spectra of the prion-like domain at 25°C in 1 mM phosphate buffer at pH 6.8 after 15 min (blue) and after 24 hr (red).

Fig 2

Fig 2. NMR characterization of the self-association.

One-dimensional 1H NMR spectra over 0.6–0.96 ppm at different time points acquired at 25°C for the wild type (A), A315E (B), Q331K (C), and M337V (D), at a protein concentration of 40 μM in 1 mM phosphate buffer (pH 6.8). Stars are used to indicate the up-field NMR peaks manifested during the self-association only by the wild-type prion-like domain.

Fig 3

Fig 3. CD and fluorescence characterization of the self-association.

Far-UV CD spectra acquired at 25°C at different time points of the incubation for the wild type (A), A315E (B), Q331K (C), and M337V (D) at a protein concentration of 20 μM in 1 mM phosphate buffer (pH 6.8). Emission spectra of the intrinsic visible fluorescence for the wild type (E), A315E (F), Q331K (G), and M337V (H) in water at pH 4.0, and in 1 mM phosphate buffer (pH 6.8) at different time points of the incubation. The wavelengths of the emission maxima are labeled for the spectra of the samples in water (pH 4.0), 5 min and 6 d after dilution into 1 mM phosphate buffer (pH 6.8). The wild type has an emission maximum very different from those of the three mutants.

Fig 4

Fig 4. Electron microscope imaging.

EM images of the samples incubated for 1 week for the wild type (A), A315E (B), Q331K (C), or M337V (D). (E) EM images of the aggregates rapidly formed by the wild type at a high protein concentration (200 μM). Upper row is images of lower magnification (scale bar of 1 μm) while lower row is of higher magnification (scale bar of 200 nm).

Fig 5

Fig 5. Residue-specific conformation and dynamics of the wild type prion-like domain in aqueous solution.

(A) Residue specific (ΔCα-ΔCβ) chemical shifts of the prion-like domain at 25°C. Green arrows are used for indicating the regions populated with nascent helical conformations while red arrows for those with extended conformations. (B) Secondary structure score obtained by analyzing chemical shifts of the prion-like domain with the SSP program. A score of +1 is for the well-formed helix while a score of -1 for the well-formed extended strand. (C) {1H}-15N heteronuclear steady-state NOE (hNOE) of the prion-like domain at 25°C. (D) Residue-specific temperature coefficients of the wild-type prion-like domain in Milli-Q water at pH 4.0 (blue), in 1 mM phosphate buffer at pH 5.0 (red) and in 1 mM phosphate buffer at pH 6.0 (cyan). (E) NOE connectivities defining secondary structures of the prion-like domain. NMR data for preparing the above figures are presented in S1 Data.

Fig 6

Fig 6. NMR characterization of the interactions with ssDNA.

One-dimensional 1H NMR spectra over 0.5–3.4 ppm at molar ratios (protein:ssDNA) of 1:0 (black), 1:0.2 (red), 1:0.5 (blue) and 1:1 (pink), as well as HSQC spectra at molar ratios of 1:0 (blue), 1:0.5 (blue), and 1:1 (green), respectively, acquired at 25°C for the wild type: (A)–(B); A315E: (C)–(D); Q331K: (E)–(F); and M337V: (G)–(H) at a protein concentration of 40 μM in 1 mM phosphate buffer (pH 5.0). Star is used to indicate the up-field NMR peaks manifested upon interacting with ssDNA only by the wild type.

Fig 7

Fig 7. Interactions of the wild-type prion-like domain with membranes.

(A) Far-UV CD spectra of the wild-type prion-like domain acquired at 25°C in Milli-Q water at pH 4.0 (black), in the presence of the DMPC/DHPC bicelle (red) and DPC micelle (blue) at a ratio of 1:200. (B) Superimposition of HSQC spectra of the prion-like domain acquired at 25°C in aqueous solution (blue) and in the presence of the DMPC/DHPC bicelle at a ratio of 1:200 (red). The assignments of the disappeared HSQC peaks are labeled. Inlet: HSQC peaks of three Trp side chains in aqueous solution (blue) and in the presence of DMPC/DHPC bicelle at a ratio of 1:200 (red). (C) Far-UV CD spectra of the prion-like domain acquired at 25°C in the presence of DPC micelle (blue) at different ratios (0–200). (D) Superimposition of HSQC spectra of the prion-like domain acquired at 25°C in aqueous solution (blue) and in the presence of DPC micelle at a ratio of 1:50 (red). (E) Superimposition of HSQC spectra of the prion-like domain acquired at 25°C in aqueous solution (blue) and in the presence of DPC micelle at a ratio of 1:100 (red). (F) Superimposition of HSQC spectra of the prion-like domain acquired at 25°C in aqueous solution (blue), in the presence of DPC micelle at a ratio of 1:100 (red) and 1:400 (green).

Fig 8

Fig 8. Residue-specific conformation of the wild-type prion-like domain in DPC micelle.

(A) Residue specific (ΔCα-ΔCβ) chemical shifts of the prion-like domain in DPC micelle (red) and in aqueous solution (blue). (B) Secondary structure scores of the prion-like domain in DPC micelle (red) and in aqueous solution (blue), which were obtained by analyzing their chemical shifts of the prion-like domain with the SSP program. (C) {1H}-15N heteronuclear steady-state NOE (hNOE) of the prion-like domain in DPC micelle (red) and in aqueous solution (blue). (D) NOE connectivities defining secondary structures of the prion-like domain in DPC micelle. (E) Ratios of HSQC peak intensities of the prion-like domain in DPC micelle with and without 10 mM gadodiamide. Red line (0.59) representing the average value plus a standard deviation is set up as a cut-off. NMR data for preparing the above figures are presented in S2 Data.

Fig 9

Fig 9. Three-dimensional structure of the membrane-interacting subdomain.

(A) The sequence of the membrane-interacting subdomain Met307-Ser347 with assigned NOEs displayed. (B) Superimposition of six lowest energy structures of the membrane-interacting subdomain. (C)–(D) The electrostatic potential surfaces of the membrane-interacting subdomain structure with different orientations. (E) Structure of residues Met311-Ala325 with long-range NOEs adopting the Ω-loop and the N-part helix. (F) Superimposition of six lowest energy structures over the Ω-loop (Met311-Asn319) with the side chains of the lowest-energy structure displayed and labelled.

Fig 10

Fig 10. Conformations of the wild type and three mutants in bicelle.

(A) Far-UV CD spectra of the wild-type and three mutant domains acquired at 25°C in the presence of DMPC/DHPC bicelle at a ratio of 1:200 after 5 min and 1 d. Superimposition of HSQC spectra acquired at 25°C in the presence of the DMPC/DHPC bicelle at a ratio of 1:200 for the wild type (blue) and mutants (red) for A315E (B), Q331K (C) and M337V (D). The mutant residues with HSQC peaks significantly shifted from those of the corresponding wild-type residues are labeled.

Fig 11

Fig 11. ALS-causing point mutations remodel the energy landscape of the self-assembly of the TDP-43 prion-like domain.

(A) Energy landscape of the self-association of the wild-type prion-like domain; and (B) its assembly of the dynamic oligomers by self-association or interacting with nucleic acids, which is characteristic of the presence of a small portion of disordered regions. (C) Energy landscape of the self-association of the ALS-causing mutants; and (D) formation of the amyloid oligomers by self-association or aggregates upon interacting with nucleic acids. The dashed arrows are used to indicate the pathways which are likely to be inaccessible.

Comment in

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Grants and funding

This study is supported by Ministry of Education of Singapore (MOE) Tier 2 Grants 2011-T2-1-096 and MOE2015-T2-1-111 to Jianxing Song. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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