LiteFold/DisProt huggingface dataset:
Protein: DP01054 (UniProt Q76LA1)
Region: residues 1	to 67
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The R68X mutant (Arg68Stop) is a peptide of 67 amino acids from the N terminus of stefin B. CD spectra have shown that the R68X peptide is not folded, in contrast to the G4R mutant, which folds like wild type.
Far-UV CD spectra (Fig. 1A) for the wt stefin B and the G4R mutant are very similar; both proteins give characteristic CD spectra for α/β secondary structure with minima at 222 nm, while the R68X mutant gives a CD spectrum characteristic of unfolded proteins with a minimum at 200 nm and low ellipticity in the 210–230 nm region.
Protein: DP01054 (UniProt Q76LA1)
Region: residues 1	to 67
Annotation: amyloid fibril formation (Biological process)
Evidence: microscopy evidence used in manual assertion
Supporting statements:
The R68X mutant showed a very high tendency to fibrillate (Fig. 8 [triangle]). Fibrils formed in 1 h without the addition of TFE (Fig. 8A1,A2 [triangle]). However, these fibrils were shorter from those grown in the presence of TFE, as judged from TEM images (Fig. 8B1,B2,C [triangle]).
Protein: DP01054 (UniProt Q76LA1)
Region: residues 1	to 67
Annotation: amyloid fibril formation (Biological process)
Evidence: fluorescence evidence used in manual assertion
Supporting statements:
For the R68X mutant, in the absence of TFE and in 3% TFE, fibril formation was very much faster (Fig. 6). No distinct lag phase could be observed, and in a few hours the whole reaction reached the plateau phase.
Protein: DP01057 (UniProt Q42512)
Region: residues 41	to 139
Annotation: disorder to order (Structural transition)
Evidence: Fourier-transform infrared spectroscopy evidence used in manual assertion
Supporting statements:
Figure S5 shows FTIR spectra of the six LEA_4 proteins in EG concentrations ranging from 0 to 12 osM. Similar to decreasing RH, increasing osmolarity induces coil-helix transitions, indicated by a shift of the Amide I maxima to higher wavenumbers.
Protein: DP01057 (UniProt Q42512)
Region: residues 41	to 139
Annotation: disorder to order (Structural transition)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
All proteins showed typical random coil spectra in dilute solution. With increasing concentrations of each of the co-solvents, the changes in the spectral shape report on the formation of ordered secondary structure. In EG (Figure S6), four out of the six proteins showed a two-state transition, indicated by the isodichroic points and the typical α-helical spectral shape with two negative maxima at 208 and 222 nm at high EG concentrations.
Protein: DP01057 (UniProt Q42512)
Region: residues 41	to 139
Annotation: disorder (Structural state)
Evidence: dynamic light scattering assay evidence used in manual assertion
Supporting statements:
Simultaneous static and dynamic light scattering (SLS/DLS) measurements were conducted to determine the apparent average single particle mass (Mapp) and the apparent average hydrodynamic radius (RS) of the proteins in solution as a function of concentration (Figure S4, [30]). Absolute molecular mass and RS were obtained by extrapolation of apparent molecular mass and RS to infinite dilution. All investigated LEA proteins are essentially monomeric under fully hydrated conditions. Compared to globular proteins, RS of all investigated LEA proteins was rather large, indicating their expanded, non-compact nature.
Protein: DP01057 (UniProt Q42512)
Region: residues 41	to 139
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
All proteins showed typical random coil spectra in dilute solution.
Protein: DP01057 (UniProt Q42512)
Region: residues 41	to 139
Annotation: response to cold (Biological process)
Evidence: fluorescence evidence used in manual assertion
Supporting statements:
Therefore, we analyzed the stability of liposomes modeling the lipid composition of inner chloroplast membranes (ICMM) after a freeze/thaw cycle and a dehydration/rehydration cycle in the ethylene glycolpresence of all six LEA_4 proteins at different LEA protein: lipid mass ratios (Figure 13).
Protein: DP01059 (UniProt Q71FK2)
Region: residues 1	to 20
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The first 20 amino acids in the N-terminal side are not included in the atomic model because the region projects outwards, and its density vanishes due to high flexibility.
Cross-references: PDB:5FN1
Protein: DP01060 (UniProt A8CDV5)
Region: residues 1	to 118
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
As expected, many peaks in the NOE spectrum of LMP2A NTD were negative, indicating an overall flexible structure.
Indeed, the chemical shift dispersion was limited to a mere 1.2 ppm, which immediately confirmed the macroscopic unfolded nature of the protein. Fortunately, the resolution of each peak and the peak dispersion along the nitrogen axis (about 25 ppm) were sufficient to achieve the peak assignment. As expected, the overall chemical shifts of the LMP2A NTD are similar to those of a random-coil structure, and the CSI results also showed that the LMP2A NTD adopts a random-coil structure in its native state.
Protein: DP01060 (UniProt A8CDV5)
Region: residues 60	to 67
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Upon the binding of the WW2 peptide, six residues (Tyr60, Glu61, Asp62, Trp65, Gly66, and Asn67) just behind the N-terminal PY motif and nine residues (Leu84, Tyr85, Leu86, Gly87, Gln89, His90, Asp94, Gly95, and Leu96) preceding to the C-terminal PY motif of the LMP2A NTD completely disappeared, which means that these residues are directly involved in WW2 domain binding.
To analyze the binding of the WW2 and WW3 peptides to the LMP2A NTD, 200 μM aliquots of chemically synthesized WW2 and WW3 peptides (WW2 and WW3 domains of human Nedd4-like ubiquitin-protein ligases AIP4) were each added to 100 μM of the LMP2A NTD, and the chemical shift changes of the LMP2A NTD were monitored in HSQC spectra (WW2:Ac-PLPPGWERRVDNMGRIYYVDHFTRTTTWQRPTLE-NH2 WW3:Ac-GPLPPGWEKRTDSNGRVYFVNHNTRITQWEDPRS-NH2).
Protein: DP01060 (UniProt A8CDV5)
Region: residues 84	to 96
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Upon the binding of the WW2 peptide, six residues (Tyr60, Glu61, Asp62, Trp65, Gly66, and Asn67) just behind the N-terminal PY motif and nine residues (Leu84, Tyr85, Leu86, Gly87, Gln89, His90, Asp94, Gly95, and Leu96) preceding to the C-terminal PY motif of the LMP2A NTD completely disappeared, which means that these residues are directly involved in WW2 domain binding.
To analyze the binding of the WW2 and WW3 peptides to the LMP2A NTD, 200 μM aliquots of chemically synthesized WW2 and WW3 peptides (WW2 and WW3 domains of human Nedd4-like ubiquitin-protein ligases AIP4) were each added to 100 μM of the LMP2A NTD, and the chemical shift changes of the LMP2A NTD were monitored in HSQC spectra (WW2:Ac-PLPPGWERRVDNMGRIYYVDHFTRTTTWQRPTLE-NH2 WW3:Ac-GPLPPGWEKRTDSNGRVYFVNHNTRITQWEDPRS-NH2).
Protein: DP01060 (UniProt A8CDV5)
Region: residues 60	to 66
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Upon WW3 peptide binding, the resonances of five residues (Tyr60, Glu61, Asp62, Trp65, and Gly66) just behind the N-terminal PY motif of the LMP2A NTD disappeared, a similar situation as with WW2 binding.
To analyze the binding of the WW2 and WW3 peptides to the LMP2A NTD, 200 μM aliquots of chemically synthesized WW2 and WW3 peptides (WW2 and WW3 domains of human Nedd4-like ubiquitin-protein ligases AIP4) were each added to 100 μM of the LMP2A NTD, and the chemical shift changes of the LMP2A NTD were monitored in HSQC spectra (WW2:Ac-PLPPGWERRVDNMGRIYYVDHFTRTTTWQRPTLE-NH2 WW3:Ac-GPLPPGWEKRTDSNGRVYFVNHNTRITQWEDPRS-NH2).
Protein: DP01060 (UniProt A8CDV5)
Region: residues 86	to 110
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Upon WW3 peptide binding, the resonances of three residues (Leu86, Gly87, and Asp94) in region 9 completely disappeared, and the resonances of three residues (Ser108, Gln109, and His110) in region 10 showed chemical shift changes.
To analyze the binding of the WW2 and WW3 peptides to the LMP2A NTD, 200 μM aliquots of chemically synthesized WW2 and WW3 peptides (WW2 and WW3 domains of human Nedd4-like ubiquitin-protein ligases AIP4) were each added to 100 μM of the LMP2A NTD, and the chemical shift changes of the LMP2A NTD were monitored in HSQC spectra (WW2:Ac-PLPPGWERRVDNMGRIYYVDHFTRTTTWQRPTLE-NH2 WW3:Ac-GPLPPGWEKRTDSNGRVYFVNHNTRITQWEDPRS-NH2).
Protein: DP01060 (UniProt A8CDV5)
Region: residues 86	to 110
Annotation: molecular adaptor activity (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The LMP2A NTD-WW3 peptide complex.
Like the WW2 peptide, the WW3 peptide forms a complex with the LMP2A NTD, although the number of residues showing chemical shift changes was relatively smaller than that of the WW2 peptide (Fig. 3b).
Protein: DP01060 (UniProt A8CDV5)
Region: residues 60	to 66
Annotation: molecular adaptor activity (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The LMP2A NTD-WW3 peptide complex.
Like the WW2 peptide, the WW3 peptide forms a complex with the LMP2A NTD, although the number of residues showing chemical shift changes was relatively smaller than that of the WW2 peptide (Fig. 3b).
Protein: DP01060 (UniProt A8CDV5)
Region: residues 60	to 67
Annotation: molecular adaptor activity (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Two-dimensional 1H–15N HSQC spectra of the LMP2A NTD-WW2 peptide complex.
Like the WW2 peptide, the WW3 peptide forms a complex with the LMP2A NTD, although the number of residues showing chemical shift changes was relatively smaller than that of the WW2 peptide (Fig. 3b).
Protein: DP01060 (UniProt A8CDV5)
Region: residues 84	to 96
Annotation: molecular adaptor activity (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Two-dimensional 1H–15N HSQC spectra of the LMP2A NTD-WW2 peptide complex.
Like the WW2 peptide, the WW3 peptide forms a complex with the LMP2A NTD, although the number of residues showing chemical shift changes was relatively smaller than that of the WW2 peptide (Fig. 3b).
Protein: DP01060 (UniProt A8CDV5)
Region: residues 1	to 118
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Therefore, the structure of LMP2A NTD seems to be intrinsically unordered without any regular structures.
Protein: DP01063 (UniProt Q9Y6C2)
Region: residues 948	to 966
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Following the secondary structure numbering of homologous proteins (10), the latter unstructured stretch reads Tyr927–Gly945
The flexible character of the unstructured segment Tyr927–Gly945, suggested by the sharpness of the corresponding NMR signals, was confirmed by 15N{1H} NOE measurements, which exhibited local enhancements ranging between -90 and -110%, as opposed to an average value of -15% observed for all the other residues but the N- and C-terminal ones (not shown).
The region Tyr927–Gly945 was classified as flexible and disordered in every analysis performed, according to 15N{1H} NOE evidence.
The published residue boundaries needed to be shifted by +21 to confirm to the current UniProt sequence.
Cross-references: PDB:2KA3
PDB:2OII
Protein: DP01063 (UniProt Q9Y6C2)
Region: residues 953	to 966
Annotation: protein binding (Molecular function)
Evidence: deletion mutation phenotypic evidence used in manual assertion
Supporting statements:
The different point-mutated proteins and the shortest deletion mutant del(Leu932–Gly945) were then assayed in standard cell adhesion assays. Jurkat cells that express high levels of the α4β1 integrin attached in a dose-response assay very efficiently to wild type and mutated EMILIN1 gC1q except del(Gly932–Gly945), E933A, L932A/E933A, and wild type EMILIN1 gC1q denatured in 8 m urea.
The published residue boundaries needed to be shifted by +21 to confirm to the current UniProt sequence.
Protein: DP01063 (UniProt Q9Y6C2)
Region: residues 951	to 956
Annotation: protein binding (Molecular function)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
The different point-mutated proteins and the shortest deletion mutant del(Leu932–Gly945) were then assayed in standard cell adhesion assays. Jurkat cells that express high levels of the α4β1 integrin attached in a dose-response assay very efficiently to wild type and mutated EMILIN1 gC1q except del(Gly932–Gly945), E933A, L932A/E933A, and wild type EMILIN1 gC1q denatured in 8 m urea.
The published residue boundaries needed to be shifted by +21 to confirm to the current UniProt sequence.
Thus, amino acid substitution mutants revealed that the conserved residue Glu933 is essential for binding of EMILIN1 gC1q to integrin α4β1, whereas single mutations of several residues close to Glu933, do not affect the interaction between the two molecules.
Because static adhesion and haptotaxis experiments showed that cells are highly dependent on Glu933 for full functionality, we next mimicked cell flow in blood vessels to determine whether firm arrest of Jurkat T cells mediated by α4β1 was also related to residue Glu933. Under flow conditions at a shear rate of 20 s-1 almost no Jurkat cells had arrested on E933A-gC1q-coated surfaces up to 4 min of flow. In marked contrast up to 160 cells/mm2 were firmly arrested on coated wt gC1q-coated surfaces.
Protein: DP01063 (UniProt Q9Y6C2)
Region: residues 951	to 956
Annotation: molecular adaptor activity (Molecular function)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
The different point-mutated proteins and the shortest deletion mutant del(Leu932–Gly945) were then assayed in standard cell adhesion assays. Jurkat cells that express high levels of the α4β1 integrin attached in a dose-response assay very efficiently to wild type and mutated EMILIN1 gC1q except del(Gly932–Gly945), E933A, L932A/E933A, and wild type EMILIN1 gC1q denatured in 8 m urea.
Thus, amino acid substitution mutants revealed that the conserved residue Glu933 is essential for binding of EMILIN1 gC1q to integrin α4β1, whereas single mutations of several residues close to Glu933, do not affect the interaction between the two molecules.
Because static adhesion and haptotaxis experiments showed that cells are highly dependent on Glu933 for full functionality, we next mimicked cell flow in blood vessels to determine whether firm arrest of Jurkat T cells mediated by α4β1 was also related to residue Glu933. Under flow conditions at a shear rate of 20 s-1 almost no Jurkat cells had arrested on E933A-gC1q-coated surfaces up to 4 min of flow. In marked contrast up to 160 cells/mm2 were firmly arrested on coated wt gC1q-coated surfaces.
The published residue boundaries needed to be shifted by +21 to confirm to the current UniProt sequence.
Protein: DP01063 (UniProt Q9Y6C2)
Region: residues 948	to 966
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
One difficulty encountered with the resonance assignment of gC1q arose from the identification of residues belonging to the segment Tyr927-Gly945, which contains four proline residues. Detection and recognition of resonances for this proved difficult due to a relatively poor signal-to-noise ratio of the nitrogen correlation spectra and was only accomplished when the majority of other peaks had already been classified. Line broadening due to slow conformational averaging of this region of EMILIN1 gC1q could be a reason for the lack of detectable amide resonances. Noticeably, this segment has very low sequence homology with other proteins of the family because it includes part of a 12-residue insertion (from Ser940 to Ser951) that is unique to EMILIN1 and EMILIN2 gC1q domains. Two amino acids in the segment 927–945, i.e. Asn934 and Ser940, still have unassigned HN groups and nine amino acids have typical 1H and 15N random coil chemical shifts
The published residue boundaries needed to be shifted by +21 to confirm to the current UniProt sequence.
According to our data the secondary structure of the EMILIN1 gC1q domain is composed of nine β-strands and one unstructured region including residues 927–945.
15N{1H} NOE measurements were run at 11.4 T and 310 K, using two different relaxation/saturation delays, 3 and 5 s. As seen from the histogram in Fig. 4, the results indicate that, besides the N-terminal and C-terminal regions, the highest mobility is observed at segment 927–945.
Some residues belong to portions of the polypeptide chain with high mobility, according to 15 N{1H} NOE data (Fig. 4), i.e. the C-terminal segment including residues Glu991-Ala995, Val949, Ala958 and the unstructured region previously identified in fragment Tyr927-Gly945. This supports the hypothesis of the existence of a flexible loop (927–945) and a five-residue C-terminal segment that are separate from the main protein fold. It is worth noting that the segment Tyr927-Gly945 and the C-terminal pentapeptide showed typical random-coil chemical shifts and no TALOS prediction output nor slow-exchanging amide protons were available or detected for those amino acids.
Cross-references: PDB:2KA3
Protein: DP01065 (UniProt Q9NQI0)
Region: residues 1	to 236
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The narrow range of amide proton chemical shifts observed in a 1H/15N HSQC spectra of the two proteins (Figure S5A) confirmed that they are intrinsically disordered.
Here, we demonstrate that human Ddx4 and its isolated disordered N terminus spontaneously self-associate both in cells and in vitro into structures that are indistinguishable from the cellular Ddx4-organelles.
Protein: DP01065 (UniProt Q9NQI0)
Region: residues 1	to 236
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: spectrometry evidence used in manual assertion
Supporting statements:
Here, we demonstrate that human Ddx4 and its isolated disordered N terminus spontaneously self-associate both in cells and in vitro into structures that are indistinguishable from the cellular Ddx4-organelles.
The organelles are clearly partitioned from surrounding nuclear structures including chromatin domains and other native nuclear bodies (Figures 1C and S2B), a finding consistent with the liquid droplet-like nature suggested by FRAP measurements (Figure 2A).
These data reveal that the disordered termini of Ddx4 mediate self-organization into macroscopic fluxional spherical structures in live cells that can be classified as organelles, which can respond rapidly to changes in the cellular environment.
Protein: DP01066 (UniProt Q9NQI0-2)
Region: residues 1	to 204
Annotation: disorder (Structural state)
Evidence: proton-based nuclear magnetic resonance evidence used in manual assertion
Supporting statements:
The narrow range of amide proton chemical shifts observed in a 1H/15N HSQC spectra of the two proteins (Figure S5A) confirmed that they are intrinsically disordered.
Protein: DP01070 (UniProt P42568)
Region: residues 490	to 568
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
We show that the ANC1 homology domain (AHD) of AF9, one of the most common MLL translocation partners, is intrinsically disordered and recruits multiple transcription factors through coupled folding and binding.
15N-1H HSQC spectra showed extreme broadening, with only a small number of peaks with narrow 1H chemical shift dispersion and an insufficient number of peaks for the amino acids in the domain (Figure 1a). Since the interacting regions between AF4 and AF9 had been finely mapped (Srinivasan et al., 2004), we titrated a peptide derived from the AF9 interaction motif of AF4 (residues 761 to 774) into the AF9 AHD. This resulted in dramatically improved chemical shift dispersion and an increased number of observable peaks in the HSQC spectrum (Figure 1b).
These data show that the AF9 AHD is intrinsically disordered, and recruits AF4 through mutual synergistic folding (Demarest et al., 2002).
Protein: DP01070 (UniProt P42568)
Region: residues 490	to 568
Annotation: molecular adaptor activity (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Leukemia fusion target AF9 is an intrinsically disordered transcriptional regulator that recruits multiple partners via coupled folding and binding.
We show that the ANC1 homology domain (AHD) of AF9, one of the most common MLL translocation partners, is intrinsically disordered and recruits multiple transcription factors through coupled folding and binding.
We propose that AF9 functions as a signaling hub that regulates transcription through dynamic recruitment of cofactors in normal hematopoiesis and in acute leukemia.
In addition to AF4 family members, the AF9 AHD has previously been shown to interact with hPC3, BCoR and Dot1L. Having established that AF4 and AF9 interact through mutual synergistic folding, we hypothesized that other binding partners may interact with AF9 in a similar manner.
In order to investigate the other interactions with the AF9 AHD, we cloned DNA coding for the sequences derived from hPC3, Dot1L and BCoR into our coexpression vector and found that all of these co-purified with AF9. Using triple resonance based backbone assignments and 1H 15N heteronuclear NOE experiments, we were able to map the residues of these proteins involved in the interaction with the AF9 AHD. Immediately apparent is the similarity in the chemical shifts of the AF9 NH moieties observed in the HSQC spectra of these complexes. As chemical shifts are highly sensitive to structure, this similarity indicates that AF9 retains a similar fold in all of the complexes (Figure 3a-d).
Together, these data strongly suggest that AF9 retains a similar fold in each of the complexes, and that each of its binding partners share a common binding site on AF9. This is consistent with previous data showing that AF9 binding partners interact with the AF9 AHD in a mutually exclusive manner (Srinivasan et al., 2003; Yokoyama et al., 2010). It was previously proposed that where IDPs compete for binding to a common site, coupled folding and binding may allow one IDP to displace another without its prior dissociation, facilitating rapid exchange between high affinity partners (De Guzman et al., 2004). The differing functions of the AF9 binding partners suggest that the function of the AHD may be to regulate gene expression through alternate binding to its partners, and intrinsic disorder may facilitate their exchange as a function of local concentration and affinity.
The AHD recruits AF4, BCoR, Dot1L and hPC3 by coupled folding and binding
AF9 binding partners compete for binding to a common site