LiteFold/DisProt huggingface dataset:
Protein: DP01633 (UniProt P29083)
Region: residues 378	to 393
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
To characterize the precise interaction at the molecular level, we first solved a solution structure of the AC-D of hTFIIEα using NMR spectroscopy (Figure 1B and Table I). The protein has a globular structure with flexible and disordered tails, consisting of the 16 N-terminal residues (amino acids 378–393) and the 5 C-terminal residues (amino acids 435–439).
Cross-references: PDB:2RNQ
Protein: DP01633 (UniProt P29083)
Region: residues 378	to 393
Annotation: disorder to order (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
To characterize the precise interaction at the molecular level, we first solved a solution structure of the AC-D of hTFIIEα using NMR spectroscopy (Figure 1B and Table I). The protein has a globular structure with flexible and disordered tails, consisting of the 16 N-terminal residues (amino acids 378–393) and the 5 C-terminal residues (amino acids 435–439)
Although the N-terminal tail of hTFIIEα AC-D also becomes ordered upon binding to p62 PH-D, it forms a bent extended structure containing a S0 strand, but not α-helix.
In the free form, it is disordered (Figure 1B) but upon complex formation becomes fixed, forming a new S0 strand that extensively overlays the positively charged surface of p62 PH-D formed by K18, K19, K54, K60, K62 and K93 (Figure 3C).
Cross-references: PDB:2RNQ
PDB:2RNR
Protein: DP01633 (UniProt P29083)
Region: residues 378	to 393
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
In hTFIIEα AC-D, the NMR signals of E386, F387, E388, E389, V390, A391 and D392 were changed significantly upon addition of p62 PH-D (Supplementary Figure 1B) and also in p62 PH-D the NMR signals of K19, Q53, K54, I55, S56, E58, K60, A61, I63, Q64, L65, Q66, T74, T75 and F77 were changed by adding hTFIIEα AC-D (Supplementary Figure 2B).
Cross-references: PDB:2RNR
Protein: DP01635 (UniProt A0A0H2ZP82)
Region: residues 40	to 50
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Residues S40 to K50 do not adopt a well-defined structure within the ensemble, suggesting that this part of the protein is disordered. This was confirmed by the heteronuclear {1H}15N-heteronuclear NOE data (see below, Fig. 4C). Structural statistics are presented in Table 1.
Cross-references: PDB:3ZFJ
Protein: DP01637 (UniProt Q8NI22)
Region: residues 27	to 66
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Resonance assignment of MCFD2 allowed localization of the disordered region to the N-terminus of the protein, approximately residues 27–66 (sequence numbered to include the signal sequence).
The N-terminus, residues 27–66, contains very few medium- or long-range restraints and a similar situation is seen for residues 102–112, a flexible loop region.
Cross-references: PDB:2VRG
BMRB:15789
Protein: DP01637 (UniProt Q8NI22)
Region: residues 27	to 146
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The NMR spectrum of MCFD2 in calcium-free phosphate buffer at pH 6.0 is characteristic of an unfolded protein, and there is no evidence of secondary structure or of a hydrophobic core.
Protein: DP01637 (UniProt Q8NI22)
Region: residues 27	to 146
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Circular dichroism (CD) spectroscopy performed on MCFD2 in the presence and absence of calcium confirms the apparent calcium-dependent folding of the protein seen in the NMR spectrum. 
Authors show the protein is intrinsically unstructured in the absence of calcium by its large negative ellipticity at 200 nm and moderate ellipticity at 190 nm.
Protein: DP01637 (UniProt Q8NI22)
Region: residues 27	to 52
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Circular dichroism (CD) spectroscopy performed on MCFD2 in the presence and absence of calcium confirms the apparent calcium-dependent folding of the protein seen in the NMR spectrum. The CD spectrum changes drastically upon addition of calcium (Fig. 3a), and the spectrum of the Ca2+-bound form reveals a dominating contribution from α-helical secondary structure with minima at 207 and 222 nm and a change of sign from negative to positive CD at 197 nm. The calcium-dependent folding is further confirmed by comparison of the CD signal measured at 222 nm as a function of calcium concentration (Fig. 3b).
The spectra of wild-type MCFD2 show higher disorder and proportionally lower helical content, when compared to the spectra of the truncated protein. The calculated difference between the two spectra yields a profile, corresponding to the signal from the first 26 residues of the N-terminus, which is characteristic of an unstructured protein.
Protein: DP01637 (UniProt Q8NI22)
Region: residues 53	to 146
Annotation: order (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Circular dichroism (CD) spectroscopy performed on MCFD2 in the presence and absence of calcium confirms the apparent calcium-dependent folding of the protein seen in the NMR spectrum. The CD spectrum changes drastically upon addition of calcium (Fig. 3a), and the spectrum of the Ca2+-bound form reveals a dominating contribution from α-helical secondary structure with minima at 207 and 222 nm and a change of sign from negative to positive CD at 197 nm. The calcium-dependent folding is further confirmed by comparison of the CD signal measured at 222 nm as a function of calcium concentration (Fig. 3b).
The spectra of wild-type MCFD2 show higher disorder and proportionally lower helical content, when compared to the spectra of the truncated protein. The calculated difference between the two spectra yields a profile, corresponding to the signal from the first 26 residues of the N-terminus, which is characteristic of an unstructured protein.
Protein: DP01637 (UniProt Q8NI22)
Region: residues 53	to 146
Annotation: disorder to order (Structural transition)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Circular dichroism (CD) spectroscopy performed on MCFD2 in the presence and absence of calcium confirms the apparent calcium-dependent folding of the protein seen in the NMR spectrum. The CD spectrum changes drastically upon addition of calcium (Fig. 3a), and the spectrum of the Ca2+-bound form reveals a dominating contribution from α-helical secondary structure with minima at 207 and 222 nm and a change of sign from negative to positive CD at 197 nm. The calcium-dependent folding is further confirmed by comparison of the CD signal measured at 222 nm as a function of calcium concentration (Fig. 3b).
The spectra of wild-type MCFD2 show higher disorder and proportionally lower helical content, when compared to the spectra of the truncated protein. The calculated difference between the two spectra yields a profile, corresponding to the signal from the first 26 residues of the N-terminus, which is characteristic of an unstructured protein.
Protein: DP01637 (UniProt Q8NI22)
Region: residues 66	to 101
Annotation: order (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The NMR spectrum in the presence of calcium shows a marked difference, with clear indications of both secondary and tertiary structure, although some disorder evidently remains.
Resonance assignment of MCFD2 allowed localization of the disordered region to the N-terminus of the protein, approximately residues 27–66 (sequence numbered to include the signal sequence).
The N-terminus, residues 27–66, contains very few medium- or long-range restraints and a similar situation is seen for residues 102–112, a flexible loop region.
Cross-references: BMRB:15789
PDB:2VRG
Protein: DP01637 (UniProt Q8NI22)
Region: residues 113	to 146
Annotation: order (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The NMR spectrum in the presence of calcium shows a marked difference, with clear indications of both secondary and tertiary structure, although some disorder evidently remains.
Resonance assignment of MCFD2 allowed localization of the disordered region to the N-terminus of the protein, approximately residues 27–66 (sequence numbered to include the signal sequence).
The N-terminus, residues 27–66, contains very few medium- or long-range restraints and a similar situation is seen for residues 102–112, a flexible loop region.
Cross-references: BMRB:15789
PDB:2VRG
Protein: DP01637 (UniProt Q8NI22)
Region: residues 113	to 146
Annotation: disorder to order (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The NMR spectrum in the presence of calcium shows a marked difference, with clear indications of both secondary and tertiary structure, although some disorder evidently remains.
In summary, we have shown human MCFD2 to be disordered in the absence of calcium and to adopt a predominantly ordered structure on binding calcium, but with the N-terminus remaining disordered and conformational flexibility in the extended loop between the two EF-hand motifs.
Cross-references: BMRB:15789
PDB:2VRG
Protein: DP01637 (UniProt Q8NI22)
Region: residues 66	to 101
Annotation: disorder to order (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The NMR spectrum in the presence of calcium shows a marked difference, with clear indications of both secondary and tertiary structure, although some disorder evidently remains.
In summary, we have shown human MCFD2 to be disordered in the absence of calcium and to adopt a predominantly ordered structure on binding calcium, but with the N-terminus remaining disordered and conformational flexibility in the extended loop between the two EF-hand motifs.
Cross-references: BMRB:15789
PDB:2VRG
Protein: DP01637 (UniProt Q8NI22)
Region: residues 102	to 112
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The N-terminus, residues 27–66, contains very few medium- or long-range restraints and a similar situation is seen for residues 102–112, a flexible loop region.
Between the two EF hands is a less well-defined and apparently flexible loop (residues 102–112) that is significantly longer than the equivalent loop in calmodulin. Assignment of the residues in this loop was extremely difficult due to the weak, broadened nature of the observed resonances, indicating conformational flexibility and exchange, as further discussed below.
Cross-references: BMRB:15789
PDB:2VRG
Protein: DP01638 (UniProt P32776)
Region: residues 64	to 85
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Residues at the N terminus (1–4), at the C terminus (113–115), and in the flexible loop (64–85) of Tfb1PH, as well as residues at the N terminus (642–668) and at the C terminus (676–690) of Rad2 were not included in the calculation.
Cross-references: PDB:2LOX
BMRB:18229
Protein: DP01638 (UniProt P32776)
Region: residues 65	to 84
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The other difference between Tfb1 and other PH domains occurs in the loop connecting strands β6 and β7 (VL3). Tfb11-115 contains a very long (20 residues) and flexible loop that is highly basic. In fact, almost all of the amino acids in this region have 15N−1H heteronuclear NOE values in the range of 0.13−0.40 (data not shown), indicating a high degree of backbone motion on the picosecond to nanosecond time scale.
Cross-references: PDB:1Y5O
Protein: DP01638 (UniProt P32776)
Region: residues 66	to 84
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Structure of the Tfb1PH/EKLFTAD2 Complex. (A) Overlay of the 20 lowest-energy structures of the complex between Tfb1PH (blue) and EKLFTAD2 (yellow). The structures were superimposed using the backbone atoms C′, Cα, and N of residues 4–65 and 85–112 of Tfb1PH and residues 59–84 of EKLFTAD2.
Cross-references: PDB:2L2I
Protein: DP01638 (UniProt P32776)
Region: residues 64	to 84
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Residues at the N terminus (1–3), at the C terminus (113–115), and in the flexible loop (64–84) of Tfb1, as well as residues at the N terminus (45–46) and at the C terminus (57–58) of p53, were not included in the calculation.
Cross-references: PDB:2GS0
Protein: DP01638 (UniProt P32776)
Region: residues 64	to 84
Annotation: flexible linker (Disorder function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Residues at the N terminus (1–3), at the C terminus (113–115), and in the flexible loop (64–84) of Tfb1, as well as residues at the N terminus (45–46) and at the C terminus (57–58) of p53, were not included in the calculation.
Cross-references: PDB:2GS0
Protein: DP01638 (UniProt P32776)
Region: residues 65	to 84
Annotation: flexible linker (Disorder function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The other difference between Tfb1 and other PH domains occurs in the loop connecting strands β6 and β7 (VL3). Tfb11-115 contains a very long (20 residues) and flexible loop that is highly basic. In fact, almost all of the amino acids in this region have 15N−1H heteronuclear NOE values in the range of 0.13−0.40 (data not shown), indicating a high degree of backbone motion on the picosecond to nanosecond time scale.
Cross-references: PDB:1Y5O
Protein: DP01638 (UniProt P32776)
Region: residues 64	to 85
Annotation: flexible linker (Disorder function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Residues at the N terminus (1–4), at the C terminus (113–115), and in the flexible loop (64–85) of Tfb1PH, as well as residues at the N terminus (642–668) and at the C terminus (676–690) of Rad2 were not included in the calculation.
Cross-references: BMRB:18229
PDB:2LOX
Protein: DP01638 (UniProt P32776)
Region: residues 67	to 82
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The consensus structure of the yeast RNA polymerase II transcription pre-initiation complex shows this region of TFB1 lacks electron density, indicating it is disordered.
Cross-references: EMDB:12720
PDB:7O4J
Protein: DP01638 (UniProt P32776)
Region: residues 122	to 166
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The consensus structure of the yeast RNA polymerase II transcription pre-initiation complex shows this region of TFB1 lacks electron density, indicating it is disordered.
Cross-references: EMDB:12720
PDB:7O4J
Protein: DP01638 (UniProt P32776)
Region: residues 394	to 412
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The consensus structure of the yeast RNA polymerase II transcription pre-initiation complex shows this region of TFB1 lacks electron density, indicating it is disordered.
Cross-references: EMDB:12720
PDB:7O4J
Protein: DP01638 (UniProt P32776)
Region: residues 447	to 461
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The consensus structure of the yeast RNA polymerase II transcription pre-initiation complex shows this region of TFB1 lacks electron density, indicating it is disordered.
Cross-references: EMDB:12720
PDB:7O4J
Protein: DP01638 (UniProt P32776)
Region: residues 518	to 535
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The consensus structure of the yeast RNA polymerase II transcription pre-initiation complex shows this region of TFB1 lacks electron density, indicating it is disordered.
Cross-references: EMDB:12720
PDB:7O4J
Protein: DP01639 (UniProt P17778)
Region: residues 1	to 33
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
However, no electron density can be observed for the first 33 residues of YopM, suggesting that this part of the protein may be disordered.
Cross-references: PDB:1G9U
PDB:1JL5
Protein: DP01640 (UniProt Q13351)
Region: residues 51	to 90
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
EKLFTAD2 binds Tfb1PH in an elongated conformation devoid of any regular secondary structural elements (Fig. 3 A and B).
Cross-references: PDB:2L2I
Protein: DP01640 (UniProt Q13351)
Region: residues 51	to 90
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
EKLFTAD2 binds Tfb1PH in an elongated conformation devoid of any regular secondary structural elements (Fig. 3 A and B). β-strands β5–β7 and the H1 α-helix of Tfb1PH form an extensive interface with residues between Ala69 and Thr80 of EKLFTAD2 (Fig. 3 C and D), and this is consistent with chemical shift changes of 15N-EKLFTAD2 upon addition of Tfb1PH (Fig. S2).
Cross-references: PDB:2L2I
Protein: DP01640 (UniProt Q13351)
Region: residues 1	to 40
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Figure 1 about the 2D 1H-15N HSQC spectra of residues 1–40 of hEKLF shows a poor dispersion of amide proton chemical shifts, indicating this region is disordered.
Protein: DP01640 (UniProt Q13351)
Region: residues 1	to 90
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Figure 1 about the 2D 1H-15N HSQC spectra of residues 1-90 of hEKLF shows a poor dispersion of amide proton chemical shifts, indicating this region is disordered.
Protein: DP01640 (UniProt Q13351)
Region: residues 23	to 36
Annotation: ubiquitin binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
EKLFTAD1 is disordered in the complex with the exception of a 14-residue amphipathic α helix that forms between residues Thr23 and Ala36 upon binding UBI (Figure 3B). 
With the exception of the 14-residue α helix, EKLFTAD1 is disordered and this transition of a short region from a disordered to an ordered conformation is consistent with what has been observed for several other acidic TADs upon binding to other target proteins (Di Lello et al., 2006, Ferreon et al., 2009, Langlois et al., 2008, Wojciak et al., 2009).
Cross-references: PDB:2MBH
Protein: DP01640 (UniProt Q13351)
Region: residues 23	to 36
Annotation: order (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
EKLFTAD1 is disordered in the complex with the exception of a 14-residue amphipathic α helix that forms between residues Thr23 and Ala36 upon binding UBI (Figure 3B). 
With the exception of the 14-residue α helix, EKLFTAD1 is disordered and this transition of a short region from a disordered to an ordered conformation is consistent with what has been observed for several other acidic TADs upon binding to other target proteins (Di Lello et al., 2006, Ferreon et al., 2009, Langlois et al., 2008, Wojciak et al., 2009).
Cross-references: PDB:2MBH
Protein: DP01640 (UniProt Q13351)
Region: residues 23	to 36
Annotation: disorder to order (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
EKLFTAD1 is disordered in the complex with the exception of a 14-residue amphipathic α helix that forms between residues Thr23 and Ala36 upon binding UBI (Figure 3B). 
With the exception of the 14-residue α helix, EKLFTAD1 is disordered and this transition of a short region from a disordered to an ordered conformation is consistent with what has been observed for several other acidic TADs upon binding to other target proteins (Di Lello et al., 2006, Ferreon et al., 2009, Langlois et al., 2008, Wojciak et al., 2009).
Cross-references: PDB:2MBH
Protein: DP01640 (UniProt Q13351)
Region: residues 27	to 31
Annotation: ubiquitin binding (Molecular function)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
Three EKLFTAD1 point mutants (F27S, L28S, and W31S) were generated, and the binding of the variant proteins to UBI was tested by NMR chemical shift perturbation experiments. In each experiment, unlabeled UBI was incrementally added to the 15N-labeled EKLFTAD1 variants as described in the previous section. During these three titrations, we were not able to reach saturation or determine values for the dissociation constants. Furthermore, after addition of UBI to variant (F27S, L28S, and Trp31S) EKLFTAD1 proteins, the resulting 1H-15N HSQC spectrums were very similar to that of their free form (Figure S3). These results confirm that Phe27, Leu28, and Trp31 on the hydrophobic face of the EKLFTAD1 α helix are crucial for binding to UBI.
Protein: DP01640 (UniProt Q13351)
Region: residues 27	to 31
Annotation: ubiquitin-dependent protein catabolic process (Biological process)
Evidence: luciferase reporter gene assay evidence used in manual assertion
Supporting statements:
s expected, the RLuc/FLuc ratio of each construct was found to increase linearly as a function of the five different concentrations of EKLF expression vector tested (Figure 5A). Linear regression analysis of this data was used to calculate a slope indicative of the stability of each EKLF constructs. Under these assay conditions, the levels of the EKLF-RLuc protein were slightly more than 2-fold lower than both the ΔTAD-EKLF-RLuc and the F27S/L28S/W31S-EKLF proteins (Figure 5B). In addition, the levels of the ΔTAD-EKLF-RLuc and the F27S/L28S/W31S-EKLF proteins were very similar. These results are in agreement with earlier studies showing that the PEST1 region within the minimal TAD of EKLF plays a significant role in ubiquitin-mediated degradation of EKLF (Quadrini and Bieker, 2006) and further establishes that the UBI-interacting domain in EKLF plays an important role in regulating its steady-state levels.
The Minimal TAD of EKLF Functions as a “Degron” in Cells