LiteFold/DisProt huggingface dataset:
Protein: DP01070 (UniProt P42568)
Region: residues 490	to 568
Annotation: disorder to order (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
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: protein binding (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
Protein: DP01070 (UniProt P42568)
Region: residues 490	to 568
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism 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.
In order to understand the structural changes induced by peptide binding, we conducted circular dichroism (CD) experiments on the AF9 AHD alone and in complex with the AF4 peptide. Surprisingly, these showed that in isolation the AHD is almost entirely random coil with only a small amount of beta structure predicted using K2D (Andrade et al., 1993)(Figure 1c). Upon addition of the AF4 peptide, the domain undergoes a structural rearrangement to form a mixed alpha-beta structure consistent with the dramatic change 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: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
In order to understand the structural changes induced by peptide binding, we conducted circular dichroism (CD) experiments on the AF9 AHD alone and in complex with the AF4 peptide. Surprisingly, these showed that in isolation the AHD is almost entirely random coil with only a small amount of beta structure predicted using K2D (Andrade et al., 1993)(Figure 1c). Upon addition of the AF4 peptide, the domain undergoes a structural rearrangement to form a mixed alpha-beta structure consistent with the dramatic change in the HSQC spectrum (Figure 1b)." Results "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: protein binding (Molecular function)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
In order to understand the structural changes induced by peptide binding, we conducted circular dichroism (CD) experiments on the AF9 AHD alone and in complex with the AF4 peptide. Surprisingly, these showed that in isolation the AHD is almost entirely random coil with only a small amount of beta structure predicted using K2D (Andrade et al., 1993)(Figure 1c). Upon addition of the AF4 peptide, the domain undergoes a structural rearrangement to form a mixed alpha-beta structure consistent with the dramatic change in the HSQC spectrum (Figure 1b)." Results "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: disorder to order (Structural transition)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
In order to understand the structural changes induced by peptide binding, we conducted circular dichroism (CD) experiments on the AF9 AHD alone and in complex with the AF4 peptide. Surprisingly, these showed that in isolation the AHD is almost entirely random coil with only a small amount of beta structure predicted using K2D (Andrade et al., 1993)(Figure 1c). Upon addition of the AF4 peptide, the domain undergoes a structural rearrangement to form a mixed alpha-beta structure consistent with the dramatic change 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: disorder (Structural state)
Evidence: cleavage assay evidence used in manual assertion
Supporting statements:
We expressed the C-terminal 79 amino acids of AF9, which includes the minimal portion of AF9 required for oncogenic activity of an MLL-AF9 fusion protein in colony forming assays (Prasad et al., 1995) and roughly corresponds to the minimal domain observed in a clinical case of leukemia with an MLL-AF9 translocation (Mitterbauer et al., 1999). During initial efforts to express and purify the AHD, we found that it had limited solubility and that the domain was prone to proteolytic degradation.
Protein: DP01071 (UniProt O23764)
Region: residues 1	to 422
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Recombinant protein overexpressed and purified from E. coli displayed an ellipticity minimum at 200nm and a value near zero at 220nm in physiological buffers, which supports an overall disordered structure.
CDeT11-24 protein extracted from plant cells showed a CD spectrum similar to the spectrum of recombinant protein (Fig. 1C), indicating that there was no significant difference between native and recombinant proteins with regard to this property.
Protein: DP01071 (UniProt O23764)
Region: residues 1	to 422
Annotation: disorder (Structural state)
Evidence: temperature-induced protein unfolding evidence used in manual assertion
Supporting statements:
The CDeT11-24 protein remained soluble in heat-treated protein extracts of dried C. plantagineum tissues (Fig. 1A).
Protein: DP01071 (UniProt O23764)
Region: residues 1	to 422
Annotation: protein folding chaperone (Molecular function)
Evidence: enzymatic activity assay evidence used in manual assertion
Supporting statements:
In the presence of recombinant CDeT11-24 proteins, CS activity remained at more than 80% for 6His-CDeT11-24 and at 70% for CDeT11-24, even after three cycles of dehydration and rehydration. The protective effect slightly exceeded the effect caused by the disaccharide trehalose, which was used as a reference (Crowe et al., 2001).
Protein: DP01071 (UniProt O23764)
Region: residues 1	to 422
Annotation: lipid binding (Molecular function)
Evidence: bait-prey hybrid interaction evidence used in manual assertion
Supporting statements:
Lipid-binding assays revealed that CDeT11-24 is able to interact with phosphatidic acid, although electrostatic repulsion was expected due to the overall negative net charge of the protein under the tested physiological conditions.
Analysis of the truncated CDeT11-24 protein identified this region to be responsible for both activities: enzyme protection and phosphatidic acid interaction.
However, CDeT11-24 bound to the phospholipid monoester PA. Weak binding was also observed with cardiolipin, which is a phospholipid containing two phosphodiester groups.
Protein: DP01075 (UniProt P20676)
Region: residues 300	to 1076
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Far-UV CD measurements of purified FG Nups produce spectra characteristic of unfolded proteins (Fig. ​3A). Each FG Nup has an intensive minimum near 200 nm and low ellipticity at 222 nm that reflect extensive contributions of unstructured coil and a lack of α-helical structure.
Protein: DP01075 (UniProt P20676)
Region: residues 300	to 1076
Annotation: disorder (Structural state)
Evidence: Fourier-transform infrared spectroscopy evidence used in manual assertion
Supporting statements:
Because FTIR spectroscopy is more sensitive to β-sheet structure than CD, we also generated FTIR spectra (amide I region) for each FG Nup. These spectra show broad absorbance bands near 1,655 cm−1 corresponding to disordered conformations (Table 3 and Fig. 5, which is published as supporting information on the PNAS web site). Deconvolution (Fourier self-deconvolution and second derivative) and curve fitting of the FTIR spectra permitted quantitative analyses of secondary structure content, confirming that purified FG Nups are composed mostly (≈80%) of disordered structure (Table ​3; Fig. ​3B).
Protein: DP01075 (UniProt P20676)
Region: residues 300	to 1076
Annotation: disorder (Structural state)
Evidence: cleavage assay evidence used in manual assertion
Supporting statements:
In contrast, most FG Nups in nuclei are fully proteolysed within 60 min (category III) or 15 min (category IV) (Fig. ​4A). Nups localized to the nuclear basket structure of the NPC (Nup60p, Nup1p, and Nup2p) and to cytoplasmic fibrils (Nup159p and Nup42p) (30–32) are the most sensitive to proteinase K (Fig. ​4A). Some FG Nups located at the center of the NPC (Nup57p, Nup53p, and nNup145p) (30) degrade after a lag period of 10 min (category II), suggesting that these Nups also contain highly flexible structures but are not immediately accessible to the protease (Fig. ​4A).
Protein: DP01075 (UniProt P20676)
Region: residues 300	to 1076
Annotation: disorder (Structural state)
Evidence: gel-filtration evidence used in manual assertion
Supporting statements:
Instead, the experimentally determined RS values for the FG Nups compare favorably to theoretical RS values predicted for monomeric proteins in natively unfolded conformations (Table 4, which is published as supporting information on the PNAS web site, www.pnas.org) (17).
Protein: DP01076 (UniProt Q02629)
Region: residues 1	to 640
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Far-UV CD measurements of purified FG Nups produce spectra characteristic of unfolded proteins (Fig. ​3A). Each FG Nup has an intensive minimum near 200 nm and low ellipticity at 222 nm that reflect extensive contributions of unstructured coil and a lack of α-helical structure.
Protein: DP01076 (UniProt Q02629)
Region: residues 1	to 640
Annotation: disorder (Structural state)
Evidence: Fourier-transform infrared spectroscopy evidence used in manual assertion
Supporting statements:
Because FTIR spectroscopy is more sensitive to β-sheet structure than CD, we also generated FTIR spectra (amide I region) for each FG Nup. These spectra show broad absorbance bands near 1,655 cm−1 corresponding to disordered conformations (Table 3 and Fig. 5, which is published as supporting information on the PNAS web site). Deconvolution (Fourier self-deconvolution and second derivative) and curve fitting of the FTIR spectra permitted quantitative analyses of secondary structure content, confirming that purified FG Nups are composed mostly (≈80%) of disordered structure (Table ​3; Fig. ​3B).
Protein: DP01076 (UniProt Q02629)
Region: residues 1	to 640
Annotation: disorder (Structural state)
Evidence: cleavage assay evidence used in manual assertion
Supporting statements:
In contrast, most FG Nups in nuclei are fully proteolysed within 60 min (category III) or 15 min (category IV) (Fig. ​4A). Nups localized to the nuclear basket structure of the NPC (Nup60p, Nup1p, and Nup2p) and to cytoplasmic fibrils (Nup159p and Nup42p) (30–32) are the most sensitive to proteinase K (Fig. ​4A). Some FG Nups located at the center of the NPC (Nup57p, Nup53p, and nNup145p) (30) degrade after a lag period of 10 min (category II), suggesting that these Nups also contain highly flexible structures but are not immediately accessible to the protease (Fig. ​4A).
Protein: DP01076 (UniProt Q02629)
Region: residues 1	to 640
Annotation: disorder (Structural state)
Evidence: gel-filtration evidence used in manual assertion
Supporting statements:
Instead, the experimentally determined RS values for the FG Nups compare favorably to theoretical RS values predicted for monomeric proteins in natively unfolded conformations (Table 4, which is published as supporting information on the PNAS web site, www.pnas.org) (17).
Protein: DP01076 (UniProt Q02629)
Region: residues 2	to 580
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: in vitro assay evidence used in manual assertion
Supporting statements:
Remarkably, the Nup100 FG domain solution turned instantaneously turbid, pointing to a very rapid phase-separation and formation of small particles or liquid droplets, which can be easily recovered by centrifugation (Figure 1B). We tried to record a time course of this reaction (by static light scattering) but had to realise that the reaction had already reached its endpoint before we could place samples into the instrument and start the measurement (after 10–30 s).
Protein: DP01076 (UniProt Q02629)
Region: residues 2	to 580
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: dynamic light scattering assay evidence used in manual assertion
Supporting statements:
When applying dynamic light scattering (DLS) to the 10 µM Nup100 FG domain sample, we observed a very prominent particle population with diameters ranging mostly between 2 and 8 µm (Figure 1C). At a lower concentration (0.625 µM), we observed a broader main peak with 0.4–4 µm particles (accounting for ≈60% by mass), another peak with 25 nm assembly intermediates (10%) and residual monomers (30%). The remaining monomer concentration can be taken as an estimate for the critical concentration for this phase-separation, which hence should be in the range of ≈200 nM FG domain or ≈10 µM FG repeat units.
Protein: DP01076 (UniProt Q02629)
Region: residues 2	to 580
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: confocal laser scanning microscopy evidence used in manual assertion
Supporting statements:
Confocal laser-scanning microscopy (CLSM) of Nup100 FG particles, formed in the presence of 5% (vol/vol) Atto390-labeled FG domain tracers, indicated that the particle size increased with higher initial concentration (not shown). Most particles were of nearly spherical shape, whereby the evident deviations from perfect spheres indicated that the phase-separated objects represent solids rather than liquids (Figure 1D; further evidence for this assumption is provided below).
Protein: DP01076 (UniProt Q02629)
Region: residues 2	to 580
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
For that we recorded the local fluorescent intensity of the added Alexa488 FG domain tracer, determined absolute fluorophore concentrations by calibration with a series of internal Alexa488-MBP standards (Figure 1E,F), and used this number to derive an average intra-particle concentration of the Nup100 FG domain of 275 mg/ml (corresponding to 4.7 mM FG domain or 200 mM FG motifs). Further assuming a specific partial volume for the protein part of 0.73 ml/g, one can estimate that the polypeptide accounts for 20% and water (respectively buffer) for the remaining 80% of the particle volume. These numbers are well in line with the assumption that these particles indeed represent hydrogels.
Protein: DP01076 (UniProt Q02629)
Region: residues 2	to 580
Annotation: non-membrane-bounded organelle assembly (Biological process)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
Following recombinant expression (after modest induction), we resuspended the bacteria in a physiological buffer, gently disrupted the cells by lysozyme treatment, and subjected the lysate to a 10,000×g centrifugation step. We observed that the FG domains from Nup100 and Nup116 (the second S.c. Nup98 paralog) pelleted under these conditions (Figure 4A,B). 
Microscopic analysis revealed that the Nup100 and Nup116 ‘FG bodies’ were 10 µm-sized irregularly shaped particles that probably had been ‘glued’ together from smaller species during the centrifugation steps. They nearly perfectly excluded our passive permeation marker MBP-Cherry and yet allowed a very efficient intra-particle accumulation of the NTR⋅cargo complex Impβ⋅IBB-GFP, reaching again a particle:buffer partition coefficient of around 200 (Figure 4A,B).
Protein: DP01077 (UniProt P14907)
Region: residues 1	to 603
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Far-UV CD measurements of purified FG Nups produce spectra characteristic of unfolded proteins (Fig. ​3A). Each FG Nup has an intensive minimum near 200 nm and low ellipticity at 222 nm that reflect extensive contributions of unstructured coil and a lack of α-helical structure.
Protein: DP01077 (UniProt P14907)
Region: residues 1	to 603
Annotation: disorder (Structural state)
Evidence: cleavage assay evidence used in manual assertion
Supporting statements:
In contrast, most FG Nups in nuclei are fully proteolysed within 60 min (category III) or 15 min (category IV) (Fig. ​4A). Nups localized to the nuclear basket structure of the NPC (Nup60p, Nup1p, and Nup2p) and to cytoplasmic fibrils (Nup159p and Nup42p) (30–32) are the most sensitive to proteinase K (Fig. ​4A). Some FG Nups located at the center of the NPC (Nup57p, Nup53p, and nNup145p) (30) degrade after a lag period of 10 min (category II), suggesting that these Nups also contain highly flexible structures but are not immediately accessible to the protease (Fig. ​4A).
Protein: DP01077 (UniProt P14907)
Region: residues 1	to 603
Annotation: disorder (Structural state)
Evidence: Fourier-transform infrared spectroscopy evidence used in manual assertion
Supporting statements:
Because FTIR spectroscopy is more sensitive to β-sheet structure than CD, we also generated FTIR spectra (amide I region) for each FG Nup. These spectra show broad absorbance bands near 1,655 cm−1 corresponding to disordered conformations (Table 3 and Fig. 5, which is published as supporting information on the PNAS web site). Deconvolution (Fourier self-deconvolution and second derivative) and curve fitting of the FTIR spectra permitted quantitative analyses of secondary structure content, confirming that purified FG Nups are composed mostly (≈80%) of disordered structure (Table ​3; Fig. ​3B).
Protein: DP01077 (UniProt P14907)
Region: residues 1	to 603
Annotation: disorder (Structural state)
Evidence: gel-filtration evidence used in manual assertion
Supporting statements:
Instead, the experimentally determined RS values for the FG Nups compare favorably to theoretical RS values predicted for monomeric proteins in natively unfolded conformations (Table 4, which is published as supporting information on the PNAS web site, www.pnas.org) (17).
Protein: DP01077 (UniProt P14907)
Region: residues 262	to 603
Annotation: protein binding (Molecular function)
Evidence: glutathione S-transferase pull-down assay evidence used in manual assertion
Supporting statements:
Both GLFG domains (Nup100 and Nup116) and a control FxFG-containing protein (GST-Nup1, residues 423–816), bound wild-type importin-β effectively (Fig. 3,lanes 1, 5, and 9).
Protein: DP01077 (UniProt P14907)
Region: residues 496	to 607
Annotation: protein binding (Molecular function)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
Although the coordinates of Ib442 when bound to FF5, in the original Ib442-FF5 crystal form, was a more similar starting model than the IBB-bound structure, we thought it prudent to avoid the possibility of introducing bias arising from a Nup-bound Ib442 as a model. 
Cross-references: PDB:1O6O
Protein: DP01077 (UniProt P14907)
Region: residues 496	to 607
Annotation: disorder (Structural state)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
PDB structure shows this region remains disordered even in complex with Importin subunit beta-1.
Cross-references: PDB:1O6O
Protein: DP01078 (UniProt P40477)
Region: residues 441	to 881
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Far-UV CD measurements of purified FG Nups produce spectra characteristic of unfolded proteins (Fig. ​3A). Each FG Nup has an intensive minimum near 200 nm and low ellipticity at 222 nm that reflect extensive contributions of unstructured coil and a lack of α-helical structure.