LiteFold/DisProt huggingface dataset:
Protein: DP01106 (UniProt P16471)
Region: residues 547	to 598
Annotation: lipid binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Following the same strategy as for PRLR-ICDmp, POPS specificity was observed for PRLR-ICDmd using 31P-NMR (Figure 2A) and chemical shift changes obtained from 1 H-15NHSQC spectra identified an additional LID3 in PRLR-ICDmd comprising residues Cys547-His598 (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 281	to 285
Annotation: phosphorylation display site (Disorder function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Using in vitro kinase assays, we investigated whether incubation with these active kinases could phosphorylate PRLR-ICDmp and showed that Lyn, Fyn and c-Src resulted in tyrosine phosphorylation (Figure 5A, lanes 3–5), whereas Jak2 and Fgr
did not (Figure 5A, lanes 2 and 6).
By reassigning the phosphorylated state, phosphorylation-induced chemical shift changes were seen to locate to residues Thr280– Tyr290 and most pronounced around residues Thr280–Glu284, thereby strongly indicating Tyr283 to be phosphorylated by Fyn.
Protein: DP01108 (UniProt Q13148)
Region: residues 266	to 414
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Furthermore, the fact that the LLPS of this intrinsically disordered domain is controlled by just a few residues may explain the unusual droplet form of TDP-43 (9).
The HSQC showed in the article is a typical IDP spectra, but it was taken for granted and not specified.
Protein: DP01108 (UniProt Q13148)
Region: residues 266	to 414
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: microscopy evidence used in manual assertion
Supporting statements:
We also collected time-lapse micrographs from low to high and then back to low temperatures to confirm the reversibility of the process (Fig. 2C, and supporting Movie S1).
At 5 °C and a protein concentration of 20 μm, clear evidence of LLPS was only observed for the WT sample (Fig. 3A).
Protein: DP01108 (UniProt Q13148)
Region: residues 266	to 414
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: turbidity measurement evidence used in manual assertion
Supporting statements:
We measured the turbidity of the WT sample (the optical density at 600 nm, OD600 nm, Fig. 2A) and recorded micrographs of the condensates at different static temperatures (Fig. 2B) to confirm the occurrence of LLPS.
At 5 °C and a protein concentration of 20 μm, clear evidence of LLPS was only observed for the WT sample (Fig. 3A).
Protein: DP01108 (UniProt Q13148)
Region: residues 267	to 414
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The two dimensional NMR spectrum of the TDP-43 C-terminal domain serves as a fingerprint of protein structure, with a narrow range of 1H chemical shifts consistent with predominant disorder (Figure 1A).
For the majority of the domain, the difference between the observed Cα and Cβ chemical shift values and those predicted for an entirely disordered protein, δΔCα-δΔCβ, do not exceed ±0.75 ppm, consistent with intrinsic disorder.
The narrow assigned 1H-15N HSQC of TDP-43267–414 suggests the domain is primarily intrinsically disordered.
Protein: DP01108 (UniProt Q13148)
Region: residues 267	to 414
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: microscopy evidence used in manual assertion
Supporting statements:
However, upon addition of 150 mM NaCl, the domain rapidly undergoes LLPS (Figure 3A top). These assemblies appear as dynamic, high protein concentration liquid droplets that flow and fuse and show effectively complete fluorescence recovery after photobleaching (FRAP), highlighting their liquid-like character (Figure 3C and Movie S1).
Protein: DP01108 (UniProt Q13148)
Region: residues 267	to 414
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: fluorescence recovery after photobleaching evidence used in manual assertion
Supporting statements:
However, upon addition of 150 mM NaCl, the domain rapidly undergoes LLPS (Figure 3A top). These assemblies appear as dynamic, high protein concentration liquid droplets that flow and fuse and show effectively complete fluorescence recovery after photobleaching (FRAP), highlighting their liquid-like character (Figure 3C and Movie S1).
Protein: DP01108 (UniProt Q13148)
Region: residues 267	to 414
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: turbidity measurement evidence used in manual assertion
Supporting statements:
The extent of phase separation increases with increasing salt concentration as monitored by turbidity.
Protein: DP01108 (UniProt Q13148)
Region: residues 321	to 343
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: turbidity measurement evidence used in manual assertion
Supporting statements:
Interestingly, both removal of the region harboring the α-helical structure and these mutations (Δ321–343) as well as introduction of the helix-breaking A326P resulted in loss of phase separation, suggesting that the helical region is important for LLPS.
Protein: DP01108 (UniProt Q13148)
Region: residues 266	to 414
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: microscopy evidence used in manual assertion
Supporting statements:
The TDP-43266–414 variants undergo LLPS at different temperatures and have different NMR signal intensity profiles.
We observed that wild-type TDP-43266–414 showed reversible LLPS when we lowered the temperature (Fig. 2 a and b; all samples returned to the original transparency either from observing the microscope image or from its turbidity).
Protein: DP01108 (UniProt Q13148)
Region: residues 266	to 414
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: turbidity measurement evidence used in manual assertion
Supporting statements:
The TDP-43266–414 variants undergo LLPS at different temperatures and have different NMR signal intensity profiles.
We observed that wild-type TDP-43266–414 showed reversible LLPS when we lowered the temperature (Fig. 2 a and b; all samples returned to the original transparency either from observing the microscope image or from its turbidity).
Protein: DP01108 (UniProt Q13148)
Region: residues 266	to 414
Annotation: intracellular non-membrane-bounded organelle (Cellular component)
Evidence: turbidity measurement evidence used in manual assertion
Supporting statements:
We found that TDP-43266–414 turned into hydrogel even in 8 M urea when the protein concentration is high (above 500 μM).
Protein: DP01108 (UniProt Q13148)
Region: residues 263	to 414
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
As judged by its far-ultraviolet (UV) CD spectrum with the maximal negative signal at 199 nm and no positive signal at 190 nm (Fig 1B), it appears to be highly disordered without any stable secondary structure.
Protein: DP01108 (UniProt Q13148)
Region: residues 263	to 414
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Moreover, it has a 1H-15N heteronuclear single quantum coherence spectroscopy (HSQC) spectrum with very narrow 1H (0.86 ppm) and 15N (17.84 ppm) spectral dispersions in which three Trp residues have their sidechain HSQC peaks largely overlapped (Fig 1C). These observations indicate that it also has no tight tertiary packing.
Protein: DP01108 (UniProt Q13148)
Region: residues 263	to 414
Annotation: intracellular non-membrane-bounded organelle (Cellular component)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Interestingly, the sample, even at a concentration of 20 μM, started to self-associate in pH 6.8 buffer, as monitored by CD (Fig 1B). After 1 d, the CD signal intensity showed a slight reduction, while after 4 d, the CD spectrum changed dramatically which is similar to what have been observed on the soluble β-stranded oligomers formed by the peptides derived from the TDP-43 prion-like domain [43–45]. After 8 d, no further changes were detected and also no visible aggregate was formed. This implies that the wild-type prion-like domain is able to progressively assemble into larger but soluble oligomers at neutral pH. The deconvolution analysis of the CD spectra revealed that the soluble oligomer formed at 8 d contains ~3% helical but ~72% β-sheet and β-turn, as well as ~25% random coil conformations.
Protein: DP01108 (UniProt Q13148)
Region: residues 263	to 414
Annotation: intracellular non-membrane-bounded organelle (Cellular component)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Even after 15 min, many HSQC peaks became broadened (Fig 1D), implying the occurrence of dynamic oligomerization. After 4 hr (Fig 1E) and 9 hr (Fig 1F), most peaks became very broad and consequently the intensity became weak. After 1 d, most peaks became too broad to be detectable (Fig 1G). Furthermore, the NMR sample in pH 6.8 buffer formed hydrogels after 1 d, which could change back to solution upon shaking, similar to what was also observed on the fused in sarcoma (FUS) prion-like domain [30,49]. However, higher protein concentrations such as at 200 μM would result in the rapid precipitation with white aggregates upon dilution into the buffer at pH 6.8.
Protein: DP01108 (UniProt Q13148)
Region: residues 263	to 414
Annotation: amyloid fibril formation (Biological process)
Evidence: fluorescence evidence used in manual assertion
Supporting statements:
Interestingly, as seen in S3E Fig, immediately upon dilution into the 1 mM phosphate buffer (pH 6.8), the wild type showed a large intensity of the ThT-binding induced fluorescence with the emission maximum at ~488 nm, implying its fast formation of the β-rich amyloid-like structure. After 1 d, the intensity was further increased and then reached the highest point after 2 d, followed by the reduction of the intensity afterward.
Protein: DP01108 (UniProt Q13148)
Region: residues 263	to 414
Annotation: amyloid fibril formation (Biological process)
Evidence: microscopy evidence used in manual assertion
Supporting statements:
As seen in Fig 4A–4D, after one week, the wild type and three mutants were all able to form amyloid fibrillar structures, with the widths of fibrils ranging from 15 to 30 nm, similar to the fine structures that were detected in the neuronal TDP-43 inclusions in patients’ brain tissues [57].
Protein: DP01108 (UniProt Q13148)
Region: residues 263	to 414
Annotation: intracellular non-membrane-bounded organelle (Cellular component)
Evidence: microscopy evidence used in manual assertion
Supporting statements:
On the other hand, amorphous structures of diverse sizes were observed for the aggregates rapidly formed by diluting the wild type into 1 mM phosphate buffer at pH 6.8 to reach a concentration of 200 μM (Fig 4E).
Protein: DP01108 (UniProt Q13148)
Region: residues 263	to 414
Annotation: disorder to order (Structural transition)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Indeed, as characterized by CD spectroscopy, ssDNA was able to bind and trigger the significant conformational changes for the wild-type prion-like domain (S7A Fig).
As judged from CD spectra in the presence of DPC at different ratios (Fig 7C), gradual addition of DPC induces progressive increase of the helical conformation, with the significant transition occurring over the ratios 50–100 (prion:DPC).
Protein: DP01108 (UniProt Q13148)
Region: residues 263	to 414
Annotation: DNA binding (Molecular function)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Indeed, as characterized by CD spectroscopy, ssDNA was able to bind and trigger the significant conformational changes for the wild-type prion-like domain (S7A Fig).
Protein: DP01108 (UniProt Q13148)
Region: residues 263	to 414
Annotation: DNA binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
However, upon addition of ssDNA at a ratio of 1:0.5 (protein:ssDNA), two very up-field peaks started to manifest and their intensity become much higher at a ratio of 1:1 (Fig 6A).
Very interestingly, for the wild type, although at a ratio of 1:0.5 many peaks became too broad to be detected mostly due to the involvement in forming the large oligomer, a set of HSQC peaks is still detectable and mostly superimposable to those in the free state (Fig 6B).
Protein: DP01108 (UniProt Q13148)
Region: residues 263	to 414
Annotation: disorder to order (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
However, upon addition of ssDNA at a ratio of 1:0.5 (protein:ssDNA), two very up-field peaks started to manifest and their intensity become much higher at a ratio of 1:1 (Fig 6A).
Very interestingly, for the wild type, although at a ratio of 1:0.5 many peaks became too broad to be detected mostly due to the involvement in forming the large oligomer, a set of HSQC peaks is still detectable and mostly superimposable to those in the free state (Fig 6B).
SSP analysis and manifestation of a large amount of NOEs indicate that the membrane-interacting subdomain transforms into a well-folded structure in the membrane environment.
Furthermore, consistent with the CD results, HSQC titrations also showed that the prion-like domain underwent conformational changes with gradual addition of DPC, as indicated by the significant shifts of some HSQC peaks (Fig 7D and 7E).
Cross-references: PDB:2N2C
Protein: DP01108 (UniProt Q13148)
Region: residues 263	to 414
Annotation: lipid binding (Molecular function)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
As seen in Fig 7A, in the presence of bicelle at a ratio of 1:200 (prion:bicelle), the prion-like domain has a far-UV CD spectrum very different from that in aqueous solution, with the maximal negative signal shifted from 198 to 203 nm; and a new negative signal at 222 nm, implying that the prion-like domain indeed has the subdomain which can interact with membranes to form helical conformation.
As judged from CD spectra in the presence of DPC at different ratios (Fig 7C), gradual addition of DPC induces progressive increase of the helical conformation, with the significant transition occurring over the ratios 50–100 (prion:DPC).
Protein: DP01108 (UniProt Q13148)
Region: residues 263	to 414
Annotation: lipid binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Furthermore, in the presence of bicelle, many HSQC peaks underwent shifts and some even completely disappeared, as well as the peaks of three Trp side chains became well-separated (Fig 7B). This observation suggests that upon interacting with bicelle, each of Trp side chains has different chemical environment. Strikingly, the disappeared peaks were identified to be from residues Met307-Gln344, indicating that these residues became tightly associated with the large bicelle, consequently their HSQC peaks became too broad to be detected due to shortening of their T2 values [79].
Furthermore, consistent with the CD results, HSQC titrations also showed that the prion-like domain underwent conformational changes with gradual addition of DPC, as indicated by the significant shifts of some HSQC peaks (Fig 7D and 7E).
SSP analysis and manifestation of a large amount of NOEs indicate that the membrane-interacting subdomain transforms into a well-folded structure in the membrane environment.
Cross-references: PDB:2N2C
Protein: DP01109 (UniProt P22626)
Region: residues 190	to 341
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The two-dimensional 1H-15N NMR correlation spectra (heteronuclear single quantum coherence, HSQC) of wild type (WT), D290V, and P298L hnRNPA2 LC have a narrow chemical shift range, characteristic of a disordered protein (Figure 1A).
Protein: DP01109 (UniProt P22626)
Region: residues 190	to 341
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: turbidity measurement evidence used in manual assertion
Supporting statements:
In addition, all hnRNPA2 LC variants show initial turbidity consistent with liquid-liquid phase separation but only D290V and P298L show turbidity at time points longer than 3 hours (Figure S4A), suggesting formation of static aggregates.
Protein: DP01109 (UniProt P22626)
Region: residues 190	to 341
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: fluorescence recovery after photobleaching evidence used in manual assertion
Supporting statements:
At 50 mM NaCl, FRAP experiments indicate that droplets of WT, D290V, P298L are liquid at early time points, as the fluorescent signal redistributes after bleaching part of individual droplets (Figure 3D).
Protein: DP01109 (UniProt P22626)
Region: residues 190	to 341
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: microscopy evidence used in manual assertion
Supporting statements:
DIC micrographs show hnRNPA2 LC WT, D290V, and P298L form liquid droplets. Over time, D290V droplets seed persistent aggregates with fibrous structure. P298L forms non-spherical clusters consistent with aggregation.
Protein: DP01110 (UniProt Q9UHD9)
Region: residues 450	to 576
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
1H-15N spectra of UBQLN2 450–624 revealed a concentration of amide resonances between 8.0 – 8.5 ppm in 1H dimension, consistent with a largely disordered construct (Figure 3A).
These data demonstrate that much of UBQLN2 450–624 is disordered. However, the UBA domain exhibits significant α-helical propensity, consistent with its known structure. Residues 450–460, located in the STI1-II domain, exhibit slight α-helical propensity and are predicted to be somewhat ordered according to PONDR-FIT calculations (Figure S1A).
Using a combination of backbone amide, Cα, Cβ, and CO chemical shifts, we predicted secondary structure population using the δ2D algorithm (Figure 3D) (Camilloni et al., 2012). These calculations confirmed the UBA domain to be α-helical.
Backbone R2 relaxation rates for UBQLN2 487–624 reinforced the notion that residues 487–580 were generally intrinsically disordered; R2 rates were 2–4 s-1, similar to the intrinsically disordered region in FUS in the non-phase separated state (Burke et al., 2015).
Protein: DP01110 (UniProt Q9UHD9)
Region: residues 450	to 576
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
When cells were exposed to arsensite stress, only FL UBQLN2 and UBQLN2 450–624 formed cytoplasmic puncta (Figure 2E). Moreover, even at slightly higher expression level than FL UBQLN2 (Figure 2F), UBQLN2 450–624 formed less puncta (Figures (Figures2E,2E, ,2G),2G), consistent with its reduced propensity to undergo LLPS in vitro. We noted that a portion of exogenously-introduced UBQLN2 colocalized with SG markers, whereas some exogenous UBQLN2 formed puncta that did not colocalize with SG marker.
Protein: DP01111 (UniProt Q13501)
Region: residues 341	to 386
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The additional N-terminal residues in the longer construct (341–386) are largely clustered in a small group with a narrow range of 1H chemical shifts indicative of unstructured polypeptide chain (shown in box).
Thus, the NMR and CD data are in good agreement and suggest that the N-terminal extension in the 341–440 UBA construct is not significantly structured.
Protein: DP01111 (UniProt Q13501)
Region: residues 341	to 386
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The spectrum of the longer construct has a more pronounced minimum at 205 nm, which indicates a significant contribution to the spectrum from a disordered random coil part of the polypeptide.
Thus, the NMR and CD data are in good agreement and suggest that the N-terminal extension in the 341–440 UBA construct is not significantly structured.
Protein: DP01112 (UniProt P27986)
Region: residues 91	to 104
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
NMR analysis provides no evidence of short- or long-range NOE interactions in the free peptide.
Protein: DP01112 (UniProt P27986)
Region: residues 91	to 104
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
 The binding of the peptide to the SH3 domain was investigated using NMR, CD, and ITC at 30 °C (Table 2). Despite the quite different methods the binding data are in good agreement. Using these very different probes of binding the average KD is found to be of the order of 31 μM, giving a free energy ΔG° for the interaction of −26 kJ·mol-1.
Cross-references: PDB:1A0N
PDB:1AZG
Protein: DP01112 (UniProt P27986)
Region: residues 91	to 104
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The featureless CD spectrum is consistent with the free peptide being nonstructured.
Protein: DP01112 (UniProt P27986)
Region: residues 91	to 104
Annotation: protein binding (Molecular function)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The figure shows three independently determined spectra:  the spectrum for the free peptide at 22 °C suggesting that there is no secondary structure present; the spectrum of P2L peptide bound to the Fyn SH3 domain in a 1:1 concentration ratio with the spectrum of the free SH3 domain subtracted; the spectrum of free P2L peptide at −104 °C showing PPII helical conformation.
Protein: DP01112 (UniProt P27986)
Region: residues 91	to 104
Annotation: protein binding (Molecular function)
Evidence: isothermal titration calorimetry evidence used in manual assertion
Supporting statements:
The binding of the peptide to the SH3 domain was investigated using NMR, CD, and ITC at 30 °C (Table 2). Despite the quite different methods the binding data are in good agreement. Using these very different probes of binding the average KD is found to be of the order of 31 μM, giving a free energy ΔG° for the interaction of −26 kJ·mol-1.
Protein: DP01113 (UniProt D0PV95)
Region: residues 1	to 168
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
To test the disorder prediction of this important domain, we performed circular dichroism (CD) measurements, which demonstrate a random coil signature (32) for the isolated RGG domain compared with the predominantly alpha-helical secondary structure of the FL and terminal deletions (Fig. 5B).