LiteFold/DisProt huggingface dataset:
Protein: DP01102 (UniProt P35637)
Region: residues 508	to 526
Annotation: protein binding (Molecular function)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
The RNA-binding protein Fused in Sarcoma (FUS) undergoes LLPS in vitro and associates with condensates in cells. We show that the Importin Karyopherin-β2/Transportin-1 inhibits LLPS of FUS. This activity depends on tight binding of Karyopherin-β2 to the C-terminal proline-tyrosine nuclear localization signal (PY-NLS) of FUS.
Kapβ2 inhibits FUS turbidity and phase separation in a PY-NLS- and RanGTP-dependent manner
Cross-references: PDB:5YVG
PDB:5YVH
PDB:5YVI
Protein: DP01102 (UniProt P35637)
Region: residues 37	to 41
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The largest perturbations are observed for resonances from the segments 37SYSGY41, 97YPGY100 and 149YSPPSG154, suggesting relatively stronger binding to these elements
Amide resonances change similarly upon addition of Kapβ2 alone or Kapβ2•PY-NLS, indicating contacts outside the PY-NLS binding site of the karyopherin
Kapβ2 inhibits FUS turbidity and phase separation in a PY-NLS- and RanGTP-dependent manner
NMR analyses reveal multiple weak interactions of Kapβ2 with both folded and disordered regions across FUS.
Protein: DP01102 (UniProt P35637)
Region: residues 149	to 154
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The largest perturbations are observed for resonances from the segments 37SYSGY41, 97YPGY100 and 149YSPPSG154, suggesting relatively stronger binding to these elements
Amide resonances change similarly upon addition of Kapβ2 alone or Kapβ2•PY-NLS, indicating contacts outside the PY-NLS binding site of the karyopherin
Kapβ2 inhibits FUS turbidity and phase separation in a PY-NLS- and RanGTP-dependent manner
NMR analyses reveal multiple weak interactions of Kapβ2 with both folded and disordered regions across FUS.
Protein: DP01102 (UniProt P35637)
Region: residues 1	to 163
Annotation: intracellular non-membrane-bounded organelle (Cellular component)
Evidence: fluorescence evidence used in manual assertion
Supporting statements:
Phase-Transition of the LC Sequence Domain of FUS into a Hydrogel
By contrast, N-terminal truncations lacking the LC sequence were incapable of phase transitioning into a hydrogel-like state.
Protein: DP01102 (UniProt P35637)
Region: residues 1	to 526
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: combinatorial experimental and author inference evidence contained in single publication used in manual assertion
Supporting statements:
Full-length FUS also undergoes LLPS, but at much lower concentrations, consistent with previous reports that the RGG regions can contribute to self-association of the protein
Protein: DP01102 (UniProt P35637)
Region: residues 285	to 370
Annotation: RNA binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The 1H/15N HSQC spectrum of 17 μM 15N-FUS(164–500) shows many strong resonances with 1H chemical shifts between ~7.5 ppm and 8.5 ppm, mostly representing residues in unstructured regions of the protein, as well as numerous resonances outside of this window, which represent the folded RRM (residues 285–370) and Cys4-type ZnF (residues 421–455) domains.
Protein: DP01102 (UniProt P35637)
Region: residues 285	to 370
Annotation: disorder to order (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The 1H/15N HSQC spectrum of 17 μM 15N-FUS(164–500) shows many strong resonances with 1H chemical shifts between ~7.5 ppm and 8.5 ppm, mostly representing residues in unstructured regions of the protein, as well as numerous resonances outside of this window, which represent the folded RRM (residues 285–370) and Cys4-type ZnF (residues 421–455) domains.
Protein: DP01102 (UniProt P35637)
Region: residues 421	to 455
Annotation: disorder to order (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The 1H/15N HSQC spectrum of 17 μM 15N-FUS(164–500) shows many strong resonances with 1H chemical shifts between ~7.5 ppm and 8.5 ppm, mostly representing residues in unstructured regions of the protein, as well as numerous resonances outside of this window, which represent the folded RRM (residues 285–370) and Cys4-type ZnF (residues 421–455) domains.
Protein: DP01102 (UniProt P35637)
Region: residues 1	to 526
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: turbidity measurement evidence used in manual assertion
Supporting statements:
Removal of MBP from MBP-FUS with the Tev protease causes FUS to self-associate, producing a turbid solution (Figure 1B and S1D, E).
Protein: DP01102 (UniProt P35637)
Region: residues 1	to 163
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The lack of significant deviation between the observed shifts and those of a random coil reference demonstrate that the monomeric FUS LC domain is highly disordered, lacking significant population of α helices or β sheets (Figure S1A).
These data support our conclusion that the domain is predominantly disordered.
Because NMR chemical shifts are sensitive reporters of structure, we conclude that the LC domain retains the same disordered structure in the monomeric full-length protein as in the isolated FUS LC domain.
Protein: DP01102 (UniProt P35637)
Region: residues 1	to 163
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: turbidity measurement evidence used in manual assertion
Supporting statements:
While measuring the structural properties of the monomeric FUS LC, we noticed that our solutions of FUS turned opalescent or cloudy when placed on ice.
Protein: DP01102 (UniProt P35637)
Region: residues 1	to 526
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: turbidity measurement evidence used in manual assertion
Supporting statements:
Upon release of MBP by TEV cleavage leaving the native protein, samples as low as 1 μM full-length FUS assemble into an opalescent, phase-separated liquid (Figure 2B) resembling that formed by FUS LC. Phase separation proceeds rapidly after addition of TEV, and the extent of phase-separation is greater at higher concentrations of full-length FUS (Figure S2A).
Protein: DP01102 (UniProt P35637)
Region: residues 1	to 163
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: microscopy evidence used in manual assertion
Supporting statements:
We used differential interference contrast microscopy to visualize the formation of liquid phase-separated FUS LC (Figure 2A), which resemble FUS-containing RNP granules previously observed in cells (Bentmann et al., 2012).
Protein: DP01102 (UniProt P35637)
Region: residues 1	to 526
Annotation: intracellular non-membrane-bounded organelle (Cellular component)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
While working with concentrated forms of the recombinant, full length FUS protein linked to a GST tag (∼10mg/ml), it was noted that the protein adopted a gel-like state when stored at low temperature.
Protein: DP01102 (UniProt P35637)
Region: residues 1	to 214
Annotation: intracellular non-membrane-bounded organelle (Cellular component)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
When produced as chimeric proteins with GST, mCherry or GFP, the FUS LC domain (residues 2-214, ∼50mg/ml) yielded clear, red and green colored hydrogels respectively (Figure 3A).
Protein: DP01102 (UniProt P35637)
Region: residues 1	to 214
Annotation: amyloid fibril formation (Biological process)
Evidence: microscopy evidence used in manual assertion
Supporting statements:
To initiate morphological and structural studies of the nature of the mCherry:FUS LC hydrogel, we first gathered transmission electron microscope (TEM) images of a pre-formed hydrogel composed of either mCherry:FUS LC or a His6-tag version of the FUS LC domain (Figure 5A and 5B). Both the mCherry:FUS and His:FUS preparations revealed morphologically uniform amyloid-like fibers.
Protein: DP01102 (UniProt P35637)
Region: residues 1	to 214
Annotation: intracellular non-membrane-bounded organelle (Cellular component)
Evidence: turbidity measurement evidence used in manual assertion
Supporting statements:
When produced as chimeric proteins with GST, mCherry or GFP, the FUS LC domain (residues 2-214, ∼50mg/ml) yielded clear, red and green colored hydrogels respectively (Figure 3A).
Protein: DP01102 (UniProt P35637)
Region: residues 1	to 526
Annotation: intracellular non-membrane-bounded organelle (Cellular component)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
Whereas the control, YFP alone, was localized diffusely throughout the cytoplasm and nucleus, FUS-YFP localized to the cytoplasm where it formed numerous foci (Figure 1C).
However, FUS expression induced the formation of P-bodies and stress granules and FUS-YFP colocalized with both of these structures (Figure 2A,B).
Protein: DP01102 (UniProt P35637)
Region: residues 1	to 526
Annotation: intracellular non-membrane-bounded organelle (Cellular component)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
Upon addition of TEV protease, FUS aggregated extremely rapidly (Figure 5C). By contrast, GST-FUS remained predominantly soluble (Figure 5C). Under identical conditions neither GST nor TEV protease aggregated (Figure 5C).
Protein: DP01102 (UniProt P35637)
Region: residues 1	to 422
Annotation: intracellular non-membrane-bounded organelle (Cellular component)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
Adding back the first RGG domain (amino acids 371–422) was sufficient to restore cytoplasmic aggregation (Figure 3B, construct 1–422).
Protein: DP01102 (UniProt P35637)
Region: residues 1	to 526
Annotation: intracellular non-membrane-bounded organelle (Cellular component)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
We also noticed that FUS formed small foci in the nucleoplasm (Figure 1B).
Protein: DP01102 (UniProt P35637)
Region: residues 1	to 526
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: fluorescence recovery after photobleaching evidence used in manual assertion
Supporting statements:
The spatiotemporal analysis of such half-bleach events showed that FUS was redistributed rapidly within stress granules and nuclear FUS assemblies, from the unbleached area to the bleached area (Figures 2A and 2B; Figure S2A; Movie S2). Thus, we conclude that FUS molecules can diffuse freely in stress granules and nuclear assemblies, in agreement with a liquid material state.
Protein: DP01102 (UniProt P35637)
Region: residues 1	to 526
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
Therefore, FUS assemblies have all the hallmarks of a liquid state: they turn over quickly; are spherical; and when they fuse, they relax into one spherical assembly (Hyman et al., 2014). Taken together, these experiments show that FUS assemblies are liquid droplets, which probably form by liquid-liquid demixing in the cytoplasm or nucleoplasm.
Protein: DP01102 (UniProt P35637)
Region: residues 2	to 36
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Despite the repetitive nature of the FUS-LC sequence, we find that the structured core of FUS-LC fibrils is formed reproducibly by a specific 57-residue segment in the N-terminal half. Other segments, especially in the C-terminal half, remain dynamically disordered.
Cross-references: PDB:5W3N
Protein: DP01102 (UniProt P35637)
Region: residues 98	to 214
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Despite the repetitive nature of the FUS-LC sequence, we find that the structured core of FUS-LC fibrils is formed reproducibly by a specific 57-residue segment in the N-terminal half. Other segments, especially in the C-terminal half, remain dynamically disordered.
Cross-references: PDB:5W3N
Protein: DP01102 (UniProt P35637)
Region: residues 39	to 95
Annotation: amyloid fibril formation (Biological process)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Despite the repetitive nature of the FUS-LC sequence, we find that the structured core of FUS-LC fibrils is formed reproducibly by a specific 57-residue segment in the N-terminal half. Other segments, especially in the C-terminal half, remain dynamically disordered.
We therefore conclude that residues 39–95 constitute the structurally ordered core of FUS-LC fibrils, with partial disorder or enhanced dynamics in residues 55–62 that precludes detection or unambiguous assignment of solid state NMR signals to these sites.
Cross-references: PDB:5W3N
Protein: DP01102 (UniProt P35637)
Region: residues 1	to 211
Annotation: RNA binding (Molecular function)
Evidence: electrophoretic mobility shift assay evidence used in manual assertion
Supporting statements:
Although it was expected that RBD would bind RNA, it was surprising that the
LCD domain also bound RNA given that prion-like domains are not typically thought to have RNA-binding activity.
Protein: DP01102 (UniProt P35637)
Region: residues 305	to 368
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
We found that FUS 4FL forms aberrant droplets that are substantially smaller than full-length FUS droplets, suggesting that FUS with a dysfunctional RRM cannot form proper condensates (Figure 6B). In the EMSA, 4FL still binds RNA and forms C1and C2, but not higher-order complex, similar to LCD and LCD-RGG1 (Figures 6C and S5). This suggests that the RRM is responsible for the high-order complex formation, while other regions of FUS, such as the RGG motifs or zinc finger domain, are responsible for the C1 and C2 complex formation.
Protein: DP01102 (UniProt P35637)
Region: residues 1	to 526
Annotation: nucleoplasm (Cellular component)
Evidence: confocal microscopy evidence used in manual assertion
Supporting statements:
Full-length FUS remains soluble in nucleoplasm as expected from the RNA buffering phase separation of FUS (Figures 7A and S7A)
Protein: DP01102 (UniProt P35637)
Region: residues 1	to 526
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: wide-field microscopy evidence used in manual assertion
Supporting statements:
We find that in the absence of RNA, neither LCD nor RBD form droplets up to 15 μM while full-length FUS forms droplets by at least 2.5 μM (Figures 1B and S1A).
These results agree with previous results in the absence of RNA showing that LCD and RBD alone do not phase separate up to 30 μM while RBD+LCD in trans does not phase separate until ~25 μM compared to 5 μM for full-length FUS.
Protein: DP01102 (UniProt P35637)
Region: residues 221	to 526
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: wide-field fluorescence microscopy evidence used in manual assertion
Supporting statements:
Strikingly, we find that RBD phase separates in the presence of RNA as readily as full-length FUS, whereas LCD does not form droplets in any conditions tested with RNA (Figures 1B and ​1C).
Protein: DP01102 (UniProt P35637)
Region: residues 221	to 526
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: dynamic light scattering assay evidence used in manual assertion
Supporting statements:
Dynamic light scattering (DLS), which is sensitive to very small cluster (1–100 nm diameter) and droplet (1–10 μm) formation, further confirmed our results that FUS and RBD form micron-scale condensates (both ~100 nm clusters and ~1000 nm droplets were observed) while LCD remains dilute (10 nm diameter) in the presence of RNA (Figure S1B).
Protein: DP01102 (UniProt P35637)
Region: residues 221	to 526
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: fluorescence recovery after photobleaching evidence used in manual assertion
Supporting statements:
Similar FRAP results were observed for RBD with and without LCD after bleaching the full droplet, bleaching partial droplets to observe only intra-droplet dynamics, and when the RBD was fluorescently labeled instead of the RNA molecule (Figure S2).
Protein: DP01102 (UniProt P35637)
Region: residues 221	to 526
Annotation: RNA binding (Molecular function)
Evidence: electrophoretic mobility shift assay evidence used in manual assertion
Supporting statements:
First, we demonstrate that both RBD and LCD truncations bind RNA using an Electrophoretic mobility shift assay (EMSA) gel (Figure 3).
Together, the EMSA suggests that although both FUS domains can bind U50 RNA as monomers and dimers, the ability to form high-order complexes, presumably through multivalent interactions, is driven by the RBD and may be critical for droplet formation.
Protein: DP01102 (UniProt P35637)
Region: residues 1	to 526
Annotation: RNA binding (Molecular function)
Evidence: fluorescence resonance energy transfer evidence used in manual assertion
Supporting statements:
In agreement with these previous results, we find that full-length FUS forms a highly dynamic complex with RNA at high concentration (500 nM) at which multimer binding is favored (Figure 4B). RBD, LCD, and R + L also yield a mix of static high-FRET and dynamic mid-FRET signal (Figure 4).
These data show that both domains are critical for maintaining a dynamic FUS-RNA interaction.
Protein: DP01102 (UniProt P35637)
Region: residues 221	to 526
Annotation: RNA binding (Molecular function)
Evidence: fluorescence resonance energy transfer evidence used in manual assertion
Supporting statements:
Furthermore, at 10 nM concentration, RBD binding shows FRET fluctuations in the intermediate state that suggest the protein is not as stably bound prior to reaching the high-FRET state (Figure 5B, left).
Protein: DP01102 (UniProt P35637)
Region: residues 1	to 211
Annotation: RNA binding (Molecular function)
Evidence: fluorescence resonance energy transfer evidence used in manual assertion
Supporting statements:
The LCD also binds RNA, as was in the EMSA, but instead of undergoing a two-state process, it transitions directly to the high-FRET state (Figure 5C).
Protein: DP01102 (UniProt P35637)
Region: residues 221	to 526
Annotation: non-membrane-bounded organelle assembly (Biological process)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
Interestingly, RBD-FUS, which drives phase separation into large condensates in vitro, co-condenses into the nucleolus which is highly enriched with RNA (Figures 7C and S7C).
Protein: DP01106 (UniProt P16471)
Region: residues 236	to 598
Annotation: disorder (Structural state)
Evidence: small-angle X-ray scattering evidence used in manual assertion
Supporting statements:
A large negative minimum at 200 nm in the far-UV CD spectrum (Figure 1C) together with a linearly increasing Kratky plot obtained by SAXS (Figure 1D) collectively suggested that PRLR-ICDFL,His was highly disordered with a low content of secondary structure.
Protein: DP01106 (UniProt P16471)
Region: residues 236	to 598
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
A large negative minimum at 200 nm in the far-UV CD spectrum (Figure 1C) together with a linearly increasing Kratky plot obtained by SAXS (Figure 1D) collectively suggested that PRLR-ICDFL,His was highly disordered with a low
content of secondary structure.
Protein: DP01106 (UniProt P16471)
Region: residues 236	to 598
Annotation: disorder (Structural state)
Evidence: heteronuclear single quantum coherence spectroscopy evidence used in manual assertion
Supporting statements:
Furthermore, the 1 H-15N-HSQC spectrum of PRLR-ICDFL,His obtained by NMR spectroscopy (Figure 1E) showed low dispersion of signals in the proton dimension, characteristic of IDPs [39] and similar to data obtained for gp130 [10].
Protein: DP01106 (UniProt P16471)
Region: residues 236	to 396
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
NMR diffusion experiments were applied to measure the hydrodynamic radius (RH) of hPRLR-ICD to 74 Å±1 Å.
Protein: DP01106 (UniProt P16471)
Region: residues 236	to 396
Annotation: lipid binding (Molecular function)
Evidence: lipid binding assay evidence used in manual assertion
Supporting statements:
First, lipid-binding specificity was assessed using lipid strips pre-dotted with different phospholipids and PRLR-ICDmp specifically recognized only negatively charged phospholipids [phosphatidylserine (PS), phosphatidylethanolamine (PE) and
phosphatidylinositol phosphates (PIPs)] (Supplementary Figure S2). These lipids are hallmarks of the inner plasma membrane leaflet [46] and, importantly, PRLR-ICDmp did not recognize phosphatidylcholine (PC; Supplementary Figure S2), which is an outer leaflet constituent [46].
As seen from Figure 2(C), addition of POPC/POPS SUV to the PRLRICDFL resulted in chemical shift changes in all three identified LIDs and thus confirmed the results obtained using the truncated variants. Moreover, since the chemical shift changes of the resolved residues of the truncated and full-length PRLR-ICD variants were similar, each LID of the PRLR-ICD appeared to associate with the lipids independently most probably due to the long disordered regions separating them.
Protein: DP01106 (UniProt P16471)
Region: residues 236	to 300
Annotation: lipid binding (Molecular function)
Evidence: heteronuclear single quantum coherence spectroscopy evidence used in manual assertion
Supporting statements:
Large confined chemical shift changes were observed for residues Glu245–His300 and the majority of signals from Gly236–Phe244 disappeared as a result of binding, thus identifying a LID1 comprising residues Gly236– His300 (Figure 2B). The chemical shifts of residues Phe350– His383 were also affected by the presence of POPC/POPS SUVs thereby defining a second LID, LID2 (Figure 2B).
As seen from Figure 2(C), addition of POPC/POPS SUV to the PRLRICDFL resulted in chemical shift changes in all three identified LIDs and thus confirmed the results obtained using the truncated variants. Moreover, since the chemical shift changes of the resolved residues of the truncated and full-length PRLR-ICD variants were similar, each LID of the PRLR-ICD appeared to associate with the lipids independently most probably due to the long disordered regions separating them.
Protein: DP01106 (UniProt P16471)
Region: residues 350	to 383
Annotation: lipid binding (Molecular function)
Evidence: heteronuclear single quantum coherence spectroscopy evidence used in manual assertion
Supporting statements:
Large confined chemical shift changes were observed for residues Glu245–His300 and the majority of signals from Gly236–Phe244 disappeared as a result of binding, thus identifying a LID1 comprising residues Gly236– His300 (Figure 2B). The chemical shifts of residues Phe350– His383 were also affected by the presence of POPC/POPS SUVs thereby defining a second LID, LID2 (Figure 2B).