LiteFold/DisProt huggingface dataset:
Protein: DP01472 (UniProt Q6GX35)
Region: residues 726	to 825
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The 2D [1H,15N]-HSQC spectrum
of 15N-labelled Tarp726–825, a construct containing the G-actin binding domain (Fig. 1), shows extremely narrow
spectral dispersion in the 1H dimension of the backbone amide chemical shifts (majority of peaks within
~0.7 ppm). This is a typical characteristic of an intrinsically disordered protein (IDP).
Cross-references: BMRB:27263
Protein: DP01472 (UniProt Q6GX35)
Region: residues 726	to 825
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Tarp binds G-actin with high affinity and increases in helical content
Protein: DP01472 (UniProt Q6GX35)
Region: residues 726	to 825
Annotation: disorder to order (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Furthermore, NMR experiments in the presence of G-actin indicate this interaction affects the proposed WH2-like α-helical region, supporting results from in silico docking calculations which suggest that, when folded, this α-helix binds within the actin hydrophobic cleft as seen for other actin-associated proteins.
Protein: DP01472 (UniProt Q6GX35)
Region: residues 726	to 825
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The SRCD spectrum of Tarp726–825 (Supplementary Fig. S4) is also consistent with a secondary structure that is primarily instrinsically disordered. Furthermore, thermal-melt SRCD studies of the construct also show behaviour typical of an IDP: the spectral changes (increase in the 224 nm shoulder and decrease in the peak around 190 nm, Supplementary Fig. S4) correspond to a decreased calculated disordered content with increasing temperature, a behaviour that directly contrasts with that seen for most fully folded (not IDP) proteins.
Protein: DP01473 (UniProt Q96GU1)
Region: residues 20	to 130
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Taken together, in the present study we have shown using the experimental data at single residue resolution level that PAGE5, a member of GAGE family proteins, is an intrinsically highly disordered protein.
Protein: DP01474 (UniProt P06748)
Region: residues 189	to 259
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
His-B23.1-CR1 showed a typical random-coil pattern peaking at 195 nm (blue line), as previously reported (25).
Protein: DP01474 (UniProt P06748)
Region: residues 120	to 130
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Above the phase separation threshold (>3:1 rpL5:N130), resonances for residues within the N130 core broadened beyond detection in 2D 1H-15N TROSY spectra but not those for residues within the A2 tract (Figure 4a). Chemical shift values indicated that these residues remained disordered.
Since N130 consists of a folded pentameric core and disordered N- and C-termini, proper sampling of the relaxation profiles was necessary. Therefore, experiments were recorded with different delay times for resonances from the folded core and the disordered N- and C-termini.
Although the N- and C-termini remain disordered, the N-terminus exhibits increased rigidity as indicated by the changes in S2 values and whose relaxation behavior was best described using the globally determined τc that was fixed during the LS-MF analysis (Figure 4c,i).
Protein: DP01474 (UniProt P06748)
Region: residues 119	to 243
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The spectrum recorded for His-B23.1 -ΔN showed negative peaks at ∼222 and 200 nm (purple line). Because negative peaks at 222 and 208 nm and that at 200 nm are indicative of α-helix and random-coil structure, respectively, we inferred that His-B23.1 -ΔN contained both α-helix and random-coil structures. Consistent with previous nuclear magnetic resonance studies (24), the spectrum of B23.1-CR1.5 (amino acids 242–294) peaked at ∼222 and 208 nm (Figure 2C, light purple line). When the sequences adjacent to CTD were elongated (His-B23.1-CR and His-B23.1 -ΔN), the peak at 208 nm shifted to ∼200 nm.
These results indicated that the central region of B23 containing acidic and basic regions was disordered in solution.
Protein: DP01474 (UniProt P06748)
Region: residues 120	to 243
Annotation: rRNA binding (Molecular function)
Evidence: glutathione S-transferase pull-down assay evidence used in manual assertion
Supporting statements:
GST-B23.1 preincubated with total RNAs was recovered in the fractions yielding 28S rRNA (Figure 3C, right panel). Consistent with this result, the fractionation pattern of 28S rRNA but not of 18S rRNA shifted slightly to the bottom (Figure 3D, top graphs), suggesting that B23.1 preferentially associated with 28S rRNA. When GST-B23.1-CR1 was preincubated with RNA, its distribution pattern shifted to high-density fractions (Figure 3C, left panel, lanes 5–10). In contrast, GST-B23.1-CR1.5 was not clearly cofractionated with rRNAs.
Consistent with this result, GST pull-down assays showed that GST-B23.1-CR1 efficiently precipitated both 18S and 28S rRNA but GST-B23.1-CR1.5 and GST did so inefficiently (data not shown). These results indicated that bIDR had potential RNA binding activity and that CTD alone could not efficiently associate with rRNAs.
Protein: DP01474 (UniProt P06748)
Region: residues 120	to 188
Annotation: protein homodimerization activity (Molecular function)
Evidence: cross-linking evidence used in manual assertion
Supporting statements:
His-tagged B23.1-ΔN and B23.2-ΔN were cross-linked with 0.1% glutaraldehyde and separated using SDS–PAGE, followed by CBB staining. Both His-B23.1-ΔN and His-B23.2-ΔN were found to be monomers at low protein concentrations because of the absence of the N-terminal oligomerization domain (Figure 7E, lanes 1 and 2). Bands corresponding to dimers appeared when His-B23.1-ΔN protein concentration was increased, whereas the dimer formation of His-B23.2-ΔN was inefficient (Figure 7E, lanes 5 and 6).
Therefore, we conclude that aIDR and bIDR of both B23.1-ΔN and B23.2-ΔN interact intra-molecularly, and that B23.1-ΔN also has the potential to form inter-molecular interactions (Figure 7F).
The authors refers as aIDR and bIDR to the 120-188 and 189-243 region of Nucleophosmin, respectively.
Protein: DP01474 (UniProt P06748)
Region: residues 120	to 188
Annotation: self-inhibition (Disorder function)
Evidence: protein separation evidence used in manual assertion
Supporting statements:
The fractionation pattern of 28S and 18S rRNAs indicated that the aIDR mutants associated with both rRNAs. The fraction shifts in fractions of both rRNAs incubated with GST-B23.1-ΔA1A2 were more prominent than those of RNAs incubated with the B23.1-CR1 mutant (see Figure 3C and D), possibly due to the oligomer formation of GST-B23.1-ΔA1A2. In addition, both 28S and 18S rRNAs incubated with GST-B23.1-ΔA1A2 were recovered in higher-density fractions than those incubated with GST-B23.1-ΔA2 (Figure 5C and D).
From these results, we concluded that the RNA binding activity of bIDR is negatively regulated by aIDR and that aIDR confers RNA binding specificity on B23.1.
The authors refers as aIDR and bIDR to the 120-188 and 189-243 region of Nucleophosmin, respectively.
Protein: DP01474 (UniProt P06748)
Region: residues 189	to 243
Annotation: protein homodimerization activity (Molecular function)
Evidence: cross-linking evidence used in manual assertion
Supporting statements:
His-tagged B23.1-ΔN and B23.2-ΔN were cross-linked with 0.1% glutaraldehyde and separated using SDS–PAGE, followed by CBB staining. Both His-B23.1-ΔN and His-B23.2-ΔN were found to be monomers at low protein concentrations because of the absence of the N-terminal oligomerization domain (Figure 7E, lanes 1 and 2). Bands corresponding to dimers appeared when His-B23.1-ΔN protein concentration was increased, whereas the dimer formation of His-B23.2-ΔN was inefficient (Figure 7E, lanes 5 and 6).
Therefore, we conclude that aIDR and bIDR of both B23.1-ΔN and B23.2-ΔN interact intra-molecularly, and that B23.1-ΔN also has the potential to form inter-molecular interactions (Figure 7F).
The authors refers as aIDR and bIDR to the 120-188 and 189-243 region of Nucleophosmin, respectively.
Protein: DP01474 (UniProt P06748)
Region: residues 197	to 201
Annotation: phosphorylation display site (Disorder function)
Evidence: in vitro protein kinase assay evidence used in manual assertion
Supporting statements:
Wild-type B23.1 and histone H1 were efficiently phosphorylated by cyclin E/cdk2 purified from NIH3T3 cells (Figure 6C, lane 2), although T199A and T4A, in which known cyclin E/cdk2-target threonine residues are substituted by alanine (Figure ​6B), were not efficiently phosphorylated (Figure 6C, lanes 3 and 4). This is consistent with the previous report that T199 in B23.1 is phosphorylated by cyclin E/cdk2 (Tokuyama et al., 2001).
Protein: DP01474 (UniProt P06748)
Region: residues 217	to 221
Annotation: phosphorylation display site (Disorder function)
Evidence: in vivo protein kinase assay evidence used in manual assertion
Supporting statements:
Migration positions of Flag-tagged T199A and T219/234/237A are also shifted from those of interphase proteins, although the band shift of these mutant proteins in mitotic extracts was less than that of the wild type.
From these in vitro and in vivo observations, it is strongly suggested that at least four threonine residues, T199, T219, T234, and T237, are the potential phosphorylation targets during mitosis possibly by cyclin B/cdc2.
Protein: DP01474 (UniProt P06748)
Region: residues 233	to 237
Annotation: phosphorylation display site (Disorder function)
Evidence: in vivo protein kinase assay evidence used in manual assertion
Supporting statements:
Migration positions of Flag-tagged T199A and T219/234/237A are also shifted from those of interphase proteins, although the band shift of these mutant proteins in mitotic extracts was less than that of the wild type.
From these in vitro and in vivo observations, it is strongly suggested that at least four threonine residues, T199, T219, T234, and T237, are the potential phosphorylation targets during mitosis possibly by cyclin B/cdc2.
Protein: DP01475 (UniProt P55980)
Region: residues 877	to 1186
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The 1 H-NMR spectrum showed weak dispersion and crowded peaks of amide proton chemical shifts around the 8-ppm region. The results of the NMR study indicated that the CagA C-terminal fragment (residues 877-1186) is virtually free from a high-order structure.
Protein: DP01475 (UniProt P55980)
Region: residues 893	to 1001
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Far-UV CD spectrum of CagA-R showed a single sharp minimum at around 203 nm and a relatively low ellipticity above 210 nm, indicative of poorly structured conformations with low content of secondary structure, a feature that is often attributed to intrinsically disordered proteins of premolten globule (PMG) type.
Protein: DP01475 (UniProt P55980)
Region: residues 893	to 1001
Annotation: disorder (Structural state)
Evidence: cleavage assay evidence used in manual assertion
Supporting statements:
CagA-R was fully degraded to small peptides within 30 min of digestion, indicating that all the potential trypsin cleavage sites are exposed and, therefore, CagA-R is either fully unfolded or intrinsically disordered.
Protein: DP01475 (UniProt P55980)
Region: residues 893	to 1001
Annotation: disorder (Structural state)
Evidence: chromatography evidence used in manual assertion
Supporting statements:
Our gel filtration studies suggested that CagA-R is a PMG type intrinsically disordered protein.
Protein: DP01475 (UniProt P55980)
Region: residues 893	to 1001
Annotation: disorder (Structural state)
Evidence: temperature-induced protein unfolding evidence used in manual assertion
Supporting statements:
The forward and reverse melting curves for CagA-R were not sigmoidal, as for typical folded proteins, but progressive,
indicating no obvious structural cooperativity of unfolding. The CD spectra of CagA-R prior to heating and after cooling back to 22°C were very similar, indicating that the thermal unfolding is reversible.
Protein: DP01475 (UniProt P55980)
Region: residues 1	to 23
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The electron density map obtained from the CagA(1-876) crystal resolved the N terminus end region of CagA (residues 24-221), which corresponded to the smaller fragment of the trypsin-digested CagA N-terminal region (Figure 1A).
Cross-references: PDB:4DVY
Protein: DP01475 (UniProt P55980)
Region: residues 222	to 302
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Whereas the overall structure was defined, N-terminal CagA was characterized by the existence of short disordered stretches such as residues 222-302, 479-488, and 510-536, of which electron densities were not observed in any crystal form (Figure 4 and Table S2).
Cross-references: PDB:4DVY
Protein: DP01475 (UniProt P55980)
Region: residues 479	to 488
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Whereas the overall structure was defined, N-terminal CagA was characterized by the existence of short disordered stretches such as residues 222-302, 479-488, and 510-536, of which electron densities were not observed in any crystal form (Figure 4 and Table S2).
Cross-references: PDB:4DVY
PDB:4DVZ
Protein: DP01475 (UniProt P55980)
Region: residues 510	to 536
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Whereas the overall structure was defined, N-terminal CagA was characterized by the existence of short disordered stretches such as residues 222-302, 479-488, and 510-536, of which electron densities were not observed in any crystal form (Figure 4 and Table S2).
Cross-references: PDB:4DVY
PDB:4DVZ
Protein: DP01475 (UniProt P55980)
Region: residues 824	to 876
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
On the other hand, no electron densities were observed for residues 825-876, suggesting that these residues were structurally disordered. We thus concluded that the entire CagA protein comprises a structured N-terminal region (residues 1-829) (Table S2) and an intrinsically disordered C-terminal region (830-1186).
Cross-references: PDB:4DVY
Protein: DP01475 (UniProt P55980)
Region: residues 222	to 302
Annotation: flexible linker (Disorder function)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
Domain I, which comprises the CagA N-terminal end, is linked to Domain II by a disordered region with about 80 amino acids.
Flexible linker connecting Domain I and Domain II of CagA.
Cross-references: PDB:4DVY
Protein: DP01475 (UniProt P55980)
Region: residues 510	to 536
Annotation: flexible linker (Disorder function)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
Interestingly, the structured N-terminal CagA still contains short disordered regions such as residues 222-302, 479-488, and 510-536. Since these disordered regions are common in all crystal forms, their presence may be a unique structural property of N-terminal CagA. Such disordered regions would not only act as flexible linkers between structured portions/domains but also serve as sites for molecular interaction, modification and so on.
Flexible linker connecting Domain II and Domain III of CagA.
Cross-references: PDB:4DVY
Protein: DP01475 (UniProt P55980)
Region: residues 877	to 1186
Annotation: protein binding (Molecular function)
Evidence: co-immunoprecipitation evidence used in manual assertion
Supporting statements:
CagA(877-1186) produced in COS-7 cells or in E. coli was mixed with FLAG-tagged PAR1b expressed in COS-7 cells and the complex formation was determined by co-immunoprecipitation.
Protein: DP01475 (UniProt P55980)
Region: residues 877	to 1186
Annotation: protein binding (Molecular function)
Evidence: surface plasmon resonance evidence used in manual assertion
Supporting statements:
Using surface plasmon resonance (SPR) spectroscopy, we directly measured binding affinity of recombinant CagA to PAR1 (Figure 2A). Full-length CagA bound to PAR1b (residues 39-364) with a KD value of 3.9 ± 0.64 nM. In contrast, C-terminal CagA(877-1186) bound to PAR1b (KD = 33.0 ± 9.3 nM) with a strength that was one order of magnitude less than that of full-length CagA. Since N-terminal CagA(1-876) did not directly bind to PAR1, the result suggested the presence of a functional interplay between the N-terminal region and C-terminal region that strengthens binding of C-terminal CagA to PAR1.
Protein: DP01475 (UniProt P55980)
Region: residues 998	to 1038
Annotation: protein binding (Molecular function)
Evidence: competitive binding evidence used in manual assertion
Supporting statements:
In addition, results of a binding-competition experiment using a series of peptides derived from C-terminal CagA and a co-immunoprecipitation experiment using internal deletion mutants of CagA(877-1186) indicated that residues 998-1038 are responsible for interaction with N-terminal CagA (Figures 2D–2F).
Protein: DP01475 (UniProt P55980)
Region: residues 998	to 1038
Annotation: protein binding (Molecular function)
Evidence: co-immunoprecipitation evidence used in manual assertion
Supporting statements:
Whereas CBS contained neither an EPIYA motif nor CM sequence, a CagA mutant lacking CBS (CagAΔ998-1038) had substantially reduced SHP2-binding activity and decreased hummingbird-inducing activity in AGS cells (Figure 2H), providing additional evidence for the importance of intramolecular CagA interaction in enhancing the pathophysiological activity of C-terminal CagA.
Authors defined region 998-1038 as C-terminal binding sequence (CBS).
Protein: DP01475 (UniProt P55980)
Region: residues 877	to 1186
Annotation: molecular adaptor activity (Molecular function)
Evidence: co-immunoprecipitation evidence used in manual assertion
Supporting statements:
Domain III interacts intramolecularly with the intrinsically disordered C-terminal region, and this interaction potentiates the pathogenic scaffold/hub function of CagA.
CagA(877-1186) produced in COS-7 cells or in E. coli was mixed with FLAG-tagged PAR1b expressed in COS-7 cells and the complex formation was determined by co-immunoprecipitation.
Whereas CBS contained neither an EPIYA motif nor CM sequence, a CagA mutant lacking CBS (CagAΔ998-1038) had substantially reduced SHP2-binding activity and decreased hummingbird-inducing activity in AGS cells (Figure 2H), providing additional evidence for the importance of intramolecular CagA interaction in enhancing the pathophysiological activity of C-terminal CagA.
The C-terminal CagA(877-1186) contains the EPIYA segment and CM sequence, which respectively act as binding sites for SHP2 and PAR1 (Higashi et al., 2002b; Saadat et al., 2007). The scaffold/hub function of C-terminal CagA is responsible for the morphogenetic activity of CagA known as the hummingbird phenotype (Bagnoli et al., 2005; Higashi et al., 2005).
Protein: DP01475 (UniProt P55980)
Region: residues 877	to 1186
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
By circular dichroism (CD) spectrum analysis of E. coli-purified recombinant proteins, the N-terminal CagA(1-876) fragment gave particular CD peaks at 208 nm and 222 nm derived from α helices, and the helix content was estimated from CD spectra data to be approximately 58%, whereas the C-terminal CagA(877-1186) was virtually free of a secondary structure (Figure 6H).
Protein: DP01476 (UniProt Q02413)
Region: residues 774	to 1049
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Here, we reveal that a 276-residue DSCR construct of human desmoglein 1 is intrinsically disordered and forms an interaction hub for desmosomal proteins. In solution, it contains 6.5% helical and 10.3% beta-strand structure based on circular dichroism spectroscopy. A single monomeric state with a predominantly unfolded structure is found by size-exclusion chromatography and analytical ultracentrifugation. Thermal stability assays and nuclear magnetic resonance spectroscopy reveal a nonglobular structure under a range of solution conditions.
Results from nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy demonstrate that the DSCR of human Dsg1 is intrinsically disordered in solution, having little intrinsically stable structure but with inducible conformations that can be stabilized by co-solvents and micelles. Analysis of its hydrodynamic behavior demonstrates that the protein exists as a monomer and in an unfolded conformation rather than a globular form.
The spectrum exhibited only a single negative minimum at 198 nm, which suggests a disordered state (Fig. 5a). The estimated α-helical, β-strand, and turn content of the DSCR at 20 °C was 4.6%, 8.1%, and 7.6%, respectively, with the remaining 79.7% being assigned as disordered.
The far-UV CD spectra of the DSCR showed the typical pattern of disordered polypeptides, with little α-helical or β-strand structure.
Protein: DP01476 (UniProt Q02413)
Region: residues 774	to 1049
Annotation: disorder (Structural state)
Evidence: chromatography evidence used in manual assertion
Supporting statements:
Here, we reveal that a 276-residue DSCR construct of human desmoglein 1 is intrinsically disordered and forms an interaction hub for desmosomal proteins. In solution, it contains 6.5% helical and 10.3% beta-strand structure based on circular dichroism spectroscopy. A single monomeric state with a predominantly unfolded structure is found by size-exclusion chromatography and analytical ultracentrifugation. Thermal stability assays and nuclear magnetic resonance spectroscopy reveal a nonglobular structure under a range of solution conditions.
Based on its gel filtration elution profile, the Stokes radius (Rs) of DSCR was estimated to be 41.8 Å. This compares more favorably with the theoretical value of a fully unfolded protein of 28.6 kDa (47.6 Å) than that of a globular monomer (24.6 Å). Together, these results indicate that the DSCR has the dimensions of a largely unfolded monomer.
The DSCR protein eluted as a single peak by gel filtration with an apparent molecular mass of 121 kDa based on calibration against known molecular mass standards (Fig. 3a).
The hydrodynamic properties of the DSCR inferred by gel filtration and AUC show that the protein adopts an unfolded monomeric conformation.