LiteFold/DisProt huggingface dataset:
Protein: DP00948 (UniProt P59595)
Region: residues 366	to 382
Annotation: single-stranded DNA binding (Molecular function)
Evidence: electrophoretic mobility shift assay evidence used in manual assertion
Supporting statements:
Gel shift assay results revealed that the SARS-CoV N protein C-terminus is also able to associate with nucleic acids and residues 363-382 are the responsible interaction partner, demonstrating that this fragment might involve genomic RNA binding sites.
As shown in Figure 7, N303-422, N283-402, and N283-382 could associate with ssDNA and migrate into the gel; however, the removal of a further 20 and 40 amino acids, generating N263-362 and N243-343 (Figure 3A), caused the proteins to lose the capability of binding to ssDNA. The ability of interacting with ssDNA was restored for N343-422 (Figure 3A), as N283-422. These results thus indicated that residues 363-382 are responsible for nucleic acid binding of the SARS-CoV N protein carboxyl terminus.
Binding to DNA is used as a probe for nucleic acid binding, but the "natural" binder is RNA.
Protein: DP00949 (UniProt Q9HAU5)
Region: residues 1105	to 1129
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
To further characterize the bipartite binding region in UPF2, which interacts with UPF1, NMR spectra were recorded using two synthetic peptides, corresponding to the helical (residues 1105–1129) and β-hairpin (residues 1167–1207) motifs of UPF2, and to 15N-labelled UPF1 (115–287).
NMR measurements on the β-hairpin peptide show that it also unfolded in solution, whereas the helix peptide shows some NOEs that indicate a fractional population of helical conformation (data not shown).
Protein: DP00949 (UniProt Q9HAU5)
Region: residues 1105	to 1129
Annotation: disorder to order (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Finally, the NMR spectrum of the free 15N-UPF2(1105–1207) shows that the protein is present in an almost completely unfolded state in solution and folding is observed on addition of UPF1(115–287) (Supplementary Figure S9D).
Altogether, these data confirm that UPF2 used a disordered bipartite motif that couples UPF1 binding to folding of the two interacting elements, with the β-hairpin element having the stronger interaction.
Protein: DP00949 (UniProt Q9HAU5)
Region: residues 1105	to 1129
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
To further characterize the bipartite binding region in UPF2, which interacts with UPF1, NMR spectra were recorded using two synthetic peptides, corresponding to the helical (residues 1105–1129) and β-hairpin (residues 1167–1207) motifs of UPF2, and to 15N-labelled UPF1 (115–287). As expected, both peptides bind to different regions in UPF1 (Figure 4B and C).
On binding of the UPF2 helix peptide, residues Leu193, Ile233, Val161 and Val157 in UPF1 show large chemical-shift perturbations, consistent with the interactions seen in the crystal structure (Figure 4B).
Protein: DP00949 (UniProt Q9HAU5)
Region: residues 1167	to 1207
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
NMR spectra of the unbound peptide (residues 1167–1207) indicate that it is unstructured in solution (see below).
NMR measurements on the β-hairpin peptide show that it also unfolded in solution, whereas the helix peptide shows some NOEs that indicate a fractional population of helical conformation (data not shown).
Protein: DP00949 (UniProt Q9HAU5)
Region: residues 1167	to 1207
Annotation: disorder to order (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Finally, the NMR spectrum of the free 15N-UPF2(1105–1207) shows that the protein is present in an almost completely unfolded state in solution and folding is observed on addition of UPF1(115–287) (Supplementary Figure S9D).
Altogether, these data confirm that UPF2 used a disordered bipartite motif that couples UPF1 binding to folding of the two interacting elements, with the β-hairpin element having the stronger interaction.
Protein: DP00949 (UniProt Q9HAU5)
Region: residues 1167	to 1207
Annotation: protein binding (Molecular function)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
On the other side of the CH-domain, the UPF2 β-hairpin is inserted between loops L10 and L7, which form another hydrophobic surface involving residues Leu176, Tyr184, Phe196, Trp241, Leu242 and the region 204-VVVL-207 (Figure 2D and Supplementary Figure S7B). The main interactions involve UPF2 residues from strand βA, including Leu1174, which contacts UPF1 residues Tyr184, Val204 and Val206, and Phe1171, which interacts with Val205. Met1173 is buried in the centre of the interface, notably contacting Phe196. Residues Leu1186, Val1188, Pro1189 and Leu1194, belonging to the UPF2 strand βB, contact Trp241 of UPF1 loop L10.
Cross-references: PDB:2WJV
Protein: DP00949 (UniProt Q9HAU5)
Region: residues 1130	to 1166
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
In this structure, both the helical and β-hairpin segments of UPF2 have good and unambiguous electron density (Figure 1A, Supplementary Figure S1), although the linker between the two is only poorly defined.
Consistent with previous analyses (Meszaros et al, 2007), the disordered linker (residues 1130–1167) between the α-helical and β-hairpin of UPF2 is highly divergent in sequence and length compared with the interacting regions (Figure 2E).
Cross-references: PDB:2WJV
Protein: DP00949 (UniProt Q9HAU5)
Region: residues 1130	to 1166
Annotation: flexible linker (Disorder function)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
In this structure, both the helical and β-hairpin segments of UPF2 have good and unambiguous electron density (Figure 1A, Supplementary Figure S1), although the linker between the two is only poorly defined.
Consistent with previous analyses (Meszaros et al, 2007), the disordered linker (residues 1130–1167) between the α-helical and β-hairpin of UPF2 is highly divergent in sequence and length compared with the interacting regions (Figure 2E).
Protein: DP00949 (UniProt Q9HAU5)
Region: residues 430	to 486
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Residues 430–486 at the C-terminus of the MIF4G-1 crystallization construct are disordered in the crystal structure.
Cross-references: PDB:4CEM
Protein: DP00949 (UniProt Q9HAU5)
Region: residues 481	to 536
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Residues 481–558 following helix hB are largely disordered in the crystal structure (apart from the short helix hC) and indeed this region is highly variable between species (Supplementary Figure S2).
Cross-references: PDB:4CEK
Protein: DP00949 (UniProt Q9HAU5)
Region: residues 1025	to 1078
Annotation: disorder (Structural state)
Evidence: curator inference from database used in manual assertion
Supporting statements:
This poly-E region is inferred by the curator to be disordered, based on MobiDB-Lite and according to experimental evidence demonstrating that poly-E repeats display random-coil-like conformation (PMID:16949547)
Protein: DP00949 (UniProt Q9HAU5)
Region: residues 757	to 767
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The domains are connected via a short 11-amino acid-long linker (residues 757–767), which stretches the 23 Å distance between the C-terminus of MIF4G domain 2 and the N-terminus of MIF4G domain 3.
The dotted line represents the 10 residues linking h10 of MIF4G-2 to h1 of MIF4G-3.
Protein: DP00949 (UniProt Q9HAU5)
Region: residues 757	to 767
Annotation: flexible linker (Disorder function)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The domains are connected via a short 11-amino acid-long linker (residues 757–767), which stretches the 23 Å distance between the C-terminus of MIF4G domain 2 and the N-terminus of MIF4G domain 3.
The dotted line represents the 10 residues linking h10 of MIF4G-2 to h1 of MIF4G-3.
Protein: DP00949 (UniProt Q9HAU5)
Region: residues 1042	to 1050
Annotation: phosphorylation display site (Disorder function)
Evidence: mass spectrometry evidence used in manual assertion
Supporting statements:
Three SMG1 phosphorylation sites were identified (Supplementary Table S2): Ser886, Ser992 and Thr1042, Ser1046 or Ser1050.
Moreover, mutation of all five Ser/Thr led to a similar phosphorylation signal as the single S1046A mutant (Figure 6D), indicating that Ser1046 is the main phosphosite for SMG1 in the UPF2 MIF4G-3 construct (residues 761–1054), which we used as substrate in our assays.
Protein: DP00949 (UniProt Q9HAU5)
Region: residues 1207	to 1227
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The additional NMR signals corresponding to the last 20 residues of the larger UPF2 construct have chemical shifts that are consistent with an unstructured peptide chain (Supplementary Figure S9C).
Protein: DP00949 (UniProt Q9HAU5)
Region: residues 1167	to 1207
Annotation: protein binding (Molecular function)
Evidence: isothermal titration calorimetry evidence used in manual assertion
Supporting statements:
α-helix (1105–1129) shows the weaker binding with a Kd of 92 μM, whereas β-hairpin (1167–1207) has an ∼sixfold higher affinity with a Kd of 16 μM (Supplementary Figure S10).
Protein: DP00949 (UniProt Q9HAU5)
Region: residues 1171	to 1176
Annotation: protein binding (Molecular function)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
Mutations of β-hairpin hydrophobic residues Phe1171E, Met1173E and Leu1174E have very severe effects on the UPF1–UPF2 interaction, completely impairing UPF1 retention by UPF2. Mutation of Arg1176 (R1176E) also has an effect on complex formation. The Arg1176 side chain interacts with Tyr184 of UPF1, which, when mutated, also disrupts complex formation (Kadlec et al, 2006).
Protein: DP00949 (UniProt Q9HAU5)
Region: residues 1171	to 1176
Annotation: nuclear-transcribed mRNA catabolic process, nonsense-mediated decay (Biological process)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
In the tethering assay, mutations of residues Phe1171 and Leu1174, located in the β-hairpin region, decrease NMD efficiency between 30 and 50%, reaching ∼80% for the triple mutant, FVM1173ERE.
In the rescue assay, in which native UPF2 is siRNA depleted and the function is rescued by a transfection of wild-type or mutant UPF2 that are RNAi insensitive, we observe very little effect on NMD efficiency compared with wild-type for the α-helix mutants and a significant effect among β-hairpin mutants only for the triple mutant, FVM1173ERE, which also has the biggest loss of function in the tethering assay (Figure 5D).
In conclusion, our in vitro binding studies and in vivo functional studies show that the β-hairpin region has the dominant function in making a functional UPF2–UPF1 interaction and that the complete disruption of this interaction severely impairs NMD.
Protein: DP00949 (UniProt Q9HAU5)
Region: residues 1015	to 1054
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The PDB structure of the protein complex with UPF3A, shows this region lacks electron density, indicating it is disordered.
Cross-references: PDB:7QG6
Protein: DP00949 (UniProt Q9HAU5)
Region: residues 1105	to 1207
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Finally, the NMR spectrum of the free 15N-UPF2(1105–1207) shows that the protein is present in an almost completely unfolded state in solution and folding is observed on addition of UPF1(115–287) (Supplementary Figure S9D).
Protein: DP00949 (UniProt Q9HAU5)
Region: residues 1167	to 1207
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
To further characterize the bipartite binding region in UPF2, which interacts with UPF1, NMR spectra were recorded using two synthetic peptides, corresponding to the helical (residues 1105–1129) and β-hairpin (residues 1167–1207) motifs of UPF2, and to 15N-labelled UPF1 (115–287). As expected, both peptides bind to different regions in UPF1 (Figure 4B and C).
For the UPF2 β-hairpin peptide, UPF1 residues Val206, Val205 and Trp241 show strong chemical-shift perturbations that cluster on the corresponding binding region seen in the crystal structure (Figure 4C).
Protein: DP00949 (UniProt Q9HAU5)
Region: residues 1108	to 1128
Annotation: protein binding (Molecular function)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
The crystal structure of the UPF1(115–914)–UPF2(1105–1198) complex in the monoclinic form allows the identification of two UPF1-interacting regions of UPF2, separated by a flexible linker, in agreement with what was originally proposed for yeast UPF2 (He et al, 1996). The N-terminal part of the UPF2 (residues 1108–1128) fragment forms a long, slightly curved, amphipathic α-helix (Figure 2A).
The binding site for the UPF2 α-helix is formed by the UPF1 residues, Val157, Val161, Phe192, Leu193 and Ile233 and the aliphatic part of Arg236 belonging to loops L6 and L10 and helix α1, which create a hydrophobic surface contacting UPF2 residues, Phe1113, Ile1114, Leu1117, Met1120, Met1121 and Leu1125 (Figure 2C and Supplementary Figure S7A).
Cross-references: PDB:2WJV
Protein: DP00949 (UniProt Q9HAU5)
Region: residues 1105	to 1129
Annotation: protein binding (Molecular function)
Evidence: isothermal titration calorimetry evidence used in manual assertion
Supporting statements:
The separate helical and β-hairpin elements show different affinities for UPF1. α-helix (1105–1129) shows the weaker binding with a Kd of 92 μM, whereas β-hairpin (1167–1207) has an ∼sixfold higher affinity with a Kd of 16 μM (Supplementary Figure S10).
Protein: DP00951 (UniProt P17677)
Region: residues 1	to 238
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
As can be clearly seen from the very narrow peak dispersion in the 1H dimension of the 1H–15N HSQC spectrum (Fig. 1), GAP-43 is an intrinsically disordered protein.
Cross-references: BMRB:19246
Protein: DP00955 (UniProt P06836)
Region: residues 1	to 242
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
B50/neuromodulin is a neuronal phosphoprotein that is found in association with the inner membrane of nerve cells. In this work, we have studied the structure of bovine B50 in aqueous solution (pH 7.5) by 1H nuclear magnetic resonance (NMR) spectroscopy and our results indicate that B50 is an unstructured protein under these conditions.
In agreement with a previous study (Masure et al. 1986), B50 appears to be a largely unstructured protein without any significant amount of regular secondary structure, as indicated by NMR and CD spectroscopy.
It is obvious that there is very little chemical shift dispersion in the aliphatic region of the NMR spectrum. A two-dimensional NOESY spectrum of B50 in H20 showed that no dNN(i,i+ 1) NOE cross-peaks could be observed, indicating that B50 does not have any regular α-helical structure.
Protein: DP00955 (UniProt P06836)
Region: residues 1	to 242
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
In agreement with a previous study (Masure et al. 1986), B50 appears to be a largely unstructured protein without any significant amount of regular secondary structure, as indicated by NMR and CD spectroscopy.
The CD spectrum of B50 in 5 rnM Tris buffer gave a typical random coil spectrum for the protein (data not shown).
Protein: DP00955 (UniProt P06836)
Region: residues 38	to 51
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The interaction of a 14-residue peptide (I38-L51) encompassing the CaM-binding domain of B50 with CaM was also studied by NMR. We have found from two-dimensional transferred nuclear Overhauser enhancement experiments that the B50 peptide binds weakly to apo-CaM in an α-helical conformation; the α-helix appears to be induced by the binding of the peptide to apo-CaM.
Protein: DP00955 (UniProt P06836)
Region: residues 38	to 51
Annotation: disorder to order (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The interaction of a 14-residue peptide (I38-L51) encompassing the CaM-binding domain of B50 with CaM was also studied by NMR. We have found from two-dimensional transferred nuclear Overhauser enhancement experiments that the B50 peptide binds weakly to apo-CaM in an α-helical conformation; the α-helix appears to be induced by the binding of the peptide to apo-CaM.
Protein: DP00955 (UniProt P06836)
Region: residues 1	to 242
Annotation: localization (Biological process)
Evidence: qualitative western immunoblotting evidence used in manual assertion
Supporting statements:
We used Western blot analysis to investigate the distribution of neuromodulin and neurogranin in membrane and cytosolic extracts of adult rat brain (Fig. 1). Neuromodulin immunoreactivity was found mainly associated with the membrane fractions, but a cytosolic form of the protein also existed (Fig. 1a).
Protein: DP00955 (UniProt P06836)
Region: residues 1	to 242
Annotation: lipid binding (Molecular function)
Evidence: qualitative western immunoblotting evidence used in manual assertion
Supporting statements:
We used Western blot analysis to investigate the distribution of neuromodulin and neurogranin in membrane and cytosolic extracts of adult rat brain (Fig. 1). Neuromodulin immunoreactivity was found mainly associated with the membrane fractions, but a cytosolic form of the protein also existed (Fig. 1a).
Protein: DP00957 (UniProt C0J347)
Region: residues 264	to 464
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Consistent with this finding, the CD spectrum of the C-terminal region contains a negative peak near the 200 nm region, which is representative of unfolded proteins (Fig. 3D).
Protein: DP00957 (UniProt C0J347)
Region: residues 264	to 464
Annotation: disorder to order (Structural transition)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
 The resulting spectrum of the mix C-TcSBDS and RNA differs slightly in the 200 and 220 nm regions from the theoretical sum of the spectra from C-TcSBDS and the RNA poly-A measured separately (Fig. 7A).
A comparison of the CD spectra of the C-TcSBDS–RNA interaction with the spectrum of C-TcSBDS with 5% TFE indicates similar changes in the 200 nm region that might be due to conformation changes of C-TcSBDS following interaction with RNA.
Protein: DP00957 (UniProt C0J347)
Region: residues 264	to 464
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The conformation of the C-terminal domain was also analyzed qualitatively by NMR. As shown in Fig. 5, the 15N-HSQC NMR spectrum revealed that the majority of the resonance peaks are clustered between 8.0 ppm and 8.8 ppm in the 1H dimension, and this low dispersion is characteristic of unfolded structures [21].
Protein: DP00957 (UniProt C0J347)
Region: residues 264	to 464
Annotation: disorder (Structural state)
Evidence: cleavage assay evidence used in manual assertion
Supporting statements:
High susceptibility to proteolysis is also an indication of unfolded protein regions. Therefore, the recombinant full-length TcSBDS, N-TcSBDS and the C-terminal region C-TcSBDS were submitted to limited proteolysis. As expected, C-TcSBDS was readily digested by trypsin (Fig. 4).
Protein: DP00957 (UniProt C0J347)
Region: residues 264	to 464
Annotation: RNA binding (Molecular function)
Evidence: electrophoretic mobility shift assay evidence used in manual assertion
Supporting statements:
Interestingly, we found that the interaction of TcSBDS with RNA is mediated by the C-terminal natively unfolded region (Fig. 6A, lanes 2, 3, 6, 7).
Band shifts were observed only for the EMSA performed with the RNA poly-A probe (Fig. 6B, lanes 2 and 4) both for the full-length TcSBDS and the C-terminal natively unfolded region, C-TcSBDS, indicating that TcSBDS shows higher affinity to RNA than to DNA. EMSA assays were also performed with C-TcSBDS using synthetic RNA (Fig. 6C, lanes 1, 2–5, 6) and DNA (Fig. 6C, lanes 3, 4–7, 8) sequences, which showed results similar to those obtained with the homopolymers RNA poly-A and DNA poly-A. These data indicate that TcSBDS shows a much higher affinity to RNA than to DNA.
Protein: DP00959 (UniProt O15169)
Region: residues 295	to 500
Annotation: disorder (Structural state)
Evidence: gel-filtration evidence used in manual assertion
Supporting statements:
The large apparent size of AxinCRG295-A500 during size-exclusion chromatography could be attributed to either an unfolded extended shape or oligomerization.
Protein: DP00959 (UniProt O15169)
Region: residues 295	to 500
Annotation: disorder (Structural state)
Evidence: fluorescence evidence used in manual assertion
Supporting statements:
We monitored the fluorescence spectrum of AxinCRG295-A500 while raising the temperature from 20 °C to 80 °C (Fig. 3a and b). The fluorescence intensity of AxinCRG295-A500 peaked at similar wavelengths at minimal and maximal temperatures (Fig. 3a). In concordance, the thermal denaturation curve of AxinCRG295-A500 did not reveal a transition in fluorescence intensity (Fig. 3b).
Protein: DP00959 (UniProt O15169)
Region: residues 295	to 500
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The far-UV CD spectrum yielded a negative peak of ellipticity at 201 nm, a typical feature of random-coil structures35 (Fig. 5).
Protein: DP00959 (UniProt O15169)
Region: residues 295	to 500
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The resulting HSQC spectrum showed strongly clustered resonances in a narrow range of 7.6–8.6 ppm, which is characteristic of unfolded proteins.