A unified mechanism for LLPS of ALS/FTLD-causing FUS as well as its modulation by ATP and oligonucleic acids - PubMed (original) (raw)
A unified mechanism for LLPS of ALS/FTLD-causing FUS as well as its modulation by ATP and oligonucleic acids
Jian Kang et al. PLoS Biol. 2019.
Abstract
526-residue Fused in sarcoma (FUS) undergoes liquid-liquid phase separation (LLPS) for its functions, which can further transit into pathological aggregation. ATP and nucleic acids, the universal cellular actors, were shown to modulate LLPS of FUS in a unique manner: enhancement and then dissolution. Currently, the driving force for LLPS of FUS is still under debate, while the mechanism for the modulation remains completely undefined. Here, by NMR and differential interference contrast (DIC) imaging, we characterized conformations, dynamics, and LLPS of FUS and its domains and subsequently their molecular interactions with oligonucleic acids, including one RNA and two single-stranded DNA (ssDNA) molecules, as well as ATP, Adenosine monophosphate (AMP), and adenosine. The results reveal 1) both a prion-like domain (PLD) rich in Tyr but absent of Arg/Lys and a C-terminal domain (CTD) abundant in Arg/Lys fail to phase separate. By contrast, the entire N-terminal domain (NTD) containing the PLD and an Arg-Gly (RG)-rich region efficiently phase separate, indicating that the π-cation interaction is the major driving force; 2) despite manifesting distinctive NMR observations, ATP has been characterized to modulate LLPS by specific binding as oligonucleic acids but with much lower affinity. Our results together establish a unified mechanism in which the π-cation interaction acts as the major driving force for LLPS of FUS and also serves as the target for modulation by ATP and oligonucleic acids through specific binding. This mechanism predicts that a myriad of proteins unrelated to RNA-binding proteins (RBPs) but with Arg/Lys-rich disordered regions could be modulated by ATP and nucleic acids, thus rationalizing the pathological association of Amyotrophic lateral sclerosis (ALS)-causing C9ORF72 dipeptides with any nucleic acids to manifest cytotoxicity.
Conflict of interest statement
The authors have declared that no competing interests exist.
Figures
Fig 1. Domain organization of FUS and conformations of its NTD and CTD.
(A) Domain organization of FUS and its six differentially dissected fragments used in the present study. 526-residue FUS is composed of an N-terminal LC region (1–267), including a QGSY-rich PLD over 1–165 and RGG1 over residues 166–267; RRM, 285–370; and CTD (371–526), which contains an RG/RGG-rich region over 371–422 (RGG2), a ZnF over 423–453, and another RG/RGG-rich region over 454–526 (RGG3) carrying an NLS. (B) Two-dimensional 1H-15N NMR HSQC spectra of the 15N-labeled FUS NTD (1–267) (blue) and PLD (1–165) (red) at 20 μM in 5 mM sodium phosphate buffer at pH 6.0. (C) HSQC spectra of the 15N-labeled FUS CTD (371–526) with the ZnF folded (blue) and unfolded (red) at 20 μM in 5 mM sodium phosphate buffer at pH 6.0. CTD, C-terminal domain; FUS, Fused in sarcoma; HSQC, Heteronuclear single quantum coherence spectroscopy; LC, low sequence complexity; NLS, nuclear localization signal; NTD, N-terminal domain; PLD, prion-like domain; QGSY, Gln-Gly-Ser-Tyr; RG/RGG-rich region, Arg-Gly/Arg-Gly-Gly–rich region; RRM, RNA-recognition motif; ZnF, zinc finger.
Fig 2. NMR backbone dynamics of the FUS NTD on a ps–ns timescale.
(A) Amino-acid sequence of the FUS NTD (1–267) with the PLD (1–165) colored in purple and RGG1 (166–267) in blue. Arg residues are underlined, and one Lys residue is colored in green and underlined. (B) Spectral densities at J(0), J(ωN), and J(0.87ωH) of the 15N-labeled FUS NTD calculated from the 15N backbone relaxation data as measured at 800 MHz. FUS, Fused in sarcoma; NTD, N-terminal domain; PLD, prion-like domain; RG, Arg-Gly; RGG, Arg-Gly-Gly; RGG1, RG/RGG-rich region 1.
Fig 3. Residue-specific conformations of the FUS CTD with ZnF folded and unfolded.
Residue-specific values of the unfolded (blue) and folded (red) ZnF residues over 421–454 for (ΔCα–ΔCβ) (A), (ΔHα) (B), and SSP (C). (D) Sequence of the folded ZnF over residue Gln321–Gly354 with assigned NOE displayed. (E) Distribution of NOEs over the sequence of the folded FUS ZnF (white bars: sequential NOEs, gray bars: medium-range NOEs, and black bars: long-range NOEs). (F) Sequence alignment between the FUS ZnF and two other ZnF domains. (G) SSP values of the folded FUS ZnF (red) and Npl4 ZnF (blue). CTD, C-terminal domain; FUS, Fused in sarcoma; NOE, Nuclear Overhauser Effect; SSP, Secondary Structure Propensity; ZnF, zinc finger.
Fig 4. LLPS of FUS as modulated by ATP and nucleic acids.
(A) FUS and its domains used in the present study for characterizing the modulation of their LLPS by ATP, AMP, and adenosine, as well as one RNA and two ssDNA molecules (B). (C) DIC microscopy images of liquid droplets formed by FUS in the presence of RNA and two ssDNA at different molar ratios. The videos for outputting these images are provided in Supporting Information. AMP, Adenosine monophosphate; DIC, differential interference contrast; FUS, Fused in sarcoma; LLPS, liquid–liquid phase separation; NLS, nuclear localization signal; QGSY, Gln-Gly-Ser-Tyr; RGG-rich region, Arg-Gly-Gly–rich region; RRM, RNA-recognition motif; ssDNA, single-stranded DNA; TssDNA, telomeric ssDNA; ZnF, zinc finger.
Fig 5. NMR view of the dissolution of LLPS of the FUS NTD induced by ssDNA.
HSQC spectra of the 15N-labeled FUS NTD in the absence (blue) and in the presence of TssDNA (red) at a ratio of 1:1 (A) and at 1:2 (B). (C) HSQC spectra of the 15N-labeled FUS NTD in the presence of TssDNA at a ratio of 1:2 (blue) and at 1:5 (red). (D) HSQC spectra of the 15N-labeled FUS NTD in the absence (blue) and in the presence of TssDNA at a ratio of 1:5 (red) and of T24 at 1:5 (green). (E) Normalized HSQC peak intensity of the 15N-labeled FUS NTD in the presence of TssDNA at molar ratio of 1:0.1 (blue) and 1:2 (red) as divided by that of the FUS NTD in the free state. FUS, Fused in sarcoma; HSQC, Heteronuclear single quantum coherence spectroscopy; LLPS, liquid–liquid phase separation; NTD, N-terminal domain; ssDNA, single-stranded DNA; TssDNA, telomeric ssDNA.
Fig 6. ssDNA can displace ATP from binding the FUS NTD.
(A) HSQC spectra of the 15N-labeled FUS NTD in the absence (blue) and in the presence of ATP at 3 mM (red). (B) HSQC spectra of the 15N-labeled FUS NTD in the absence (blue), and in the presence of ATP at 3 mM with an extra addition of TssDNA at 60 μM (red). (C) HSQC spectra of the 15N-labeled FUS NTD in the presence of TssDNA at a ratio of 1:5 (blue) and in the presence of both ATP at 3 mM and TssDNA at 60 μM (red). (D) A speculative model to rationalize the specific binding of ATP to Arg/Lys residues within RGG1 of the FUS NTD, which, however, can be displaced by ssDNA. FUS, Fused in sarcoma; HSQC, Heteronuclear single quantum coherence spectroscopy; NTD, N-terminal domain; PLD, prion-like domain; RGG1, Arg-Gly-Gly–rich domain 1; ssDNA, single-stranded DNA; TssDNA, telomeric ssDNA.
Fig 7. NMR view of the induction and dissolution of LLPS of the FUS U-CTD.
(A) HSQC spectra of the 15N-labeled FUS U-CTD in the absence (blue) and in the presence of ATP at 2 mM (red). (B) HSQC spectra of the 15N-labeled FUS U-CTD in the absence (blue) and in the presence of TssDNA at a ratio of 1:10 (red). (C) HSQC spectra of the 15N-labeled FUS U-CTD in the absence (blue) and in the presence of T24 at a ratio of 1:10 (red). (D) CSD of the FUS U-CTD in the presence of ATP at 2 mM (blue) and in the presence of TssDNA (red) and T24 (green) at a ratio of 1:10. Filled circles are used for indicating the locations of Arg residues, while triangles indicate Lys. Blue is for those within RGG2, black for those in the unfolded ZnF, and purple for those within RGG3. (E) A speculative model to rationalize the specific binding of ATP and ssDNA to Arg/Lys residues within the FUS U-CTD to induce LLPS at low concentrations but to dissolve at high concentrations. CSD, chemical shift difference; CTD, C-terminal domain; FUS, Fused in sarcoma; HSQC, Heteronuclear single quantum coherence spectroscopy; LLPS, liquid–liquid phase separation; RGG, Arg-Gly/Arg-Gly-Gly–rich region; ssDNA, single-stranded DNA; TssDNA, telomeric ssDNA; U-CTD, ZnF unfolded CTD; ZnF, zinc finger.
Fig 8. NMR view of enhancement and dissolution of LLPS of FUS induced by ATP and ssDNA.
(A) Normalized HSQC peak intensity of the 15N-labeled FUS in the presence of TssDNA at molar ratios of 1: 0.1 (blue) and 1:0.5 (red) as divided by that of FUS in the free state. (B) Normalized HSQC peak intensity of the 15N-labeled FUS in the presence of TssDNA at molar ratios of 1: 0.1 (blue) and 1:10 (purple) as divided by that of FUS in the free state. (C) A speculative model to rationalize the specific binding of ATP and ssDNA to Arg/Lys residues as well as RRM and ZnF of FUS to enhance LLPS at low concentrations but dissolution at high concentrations. FUS, Fused in sarcoma; HSQC, Heteronuclear single quantum coherence spectroscopy; LLPS, liquid–liquid phase separation; PLD, prion-like domain; RGG, Arg-Gly/Arg-Gly-Gly–rich region; RRM, RNA-recognition motif; ssDNA, single-stranded DNA; TssDNA, telomeric ssDNA; ZnF, zinc finger.
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This study is supported by Ministry of Education of Singapore (MOE) Tier 2 Grant MOE2015-T2-1-111 to JS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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