LiteFold/DisProt huggingface dataset:
Protein: DP01705 (UniProt Q07666)
Region: residues 281	to 338
Annotation: RNA binding (Molecular function)
Evidence: heteronuclear single quantum coherence spectroscopy evidence used in manual assertion
Supporting statements:
Increasing the G8.5 molar ratio with the Sam68 N- and C-termini induced chemical shift perturbations, consistent with the formation of a complex between these regions and the RNA (Figure 3B, C). These results demonstrate that the Sam68 N- and C-terminal regions can bind RNA independently of the rest of the protein. Analysis of the titration data suggests that both domains bind the G.8 RNA with dissociation constants in the low micromolar range (1–10 μM for the N-terminus and 30–70 μM for the C-terminus) (Supplementary Figure S2), very similar to the affinity of the STAR domain for the same RNA sequence (18).
Analysis of the chemical shift mapping shows that RNA binding affects most residues of the N- and C-termini, in particular residues 10–61 for the N-terminus and 281–338 for the C-terminus (Figure 3D, E).
 In that case, the addition of the RNA induces only minor chemical shift perturbation, suggesting that the N- and C-terminal regions of Sam68 bind specifically to the G8.5 RNA.
G8.5 is a RNA that contains the UAAA motif and was previously identified as a high affinity Sam68 binder by SELEX.
Protein: DP01705 (UniProt Q07666)
Region: residues 31	to 35
Annotation: molecular function inhibitor activity (Molecular function)
Evidence: heteronuclear single quantum coherence spectroscopy evidence used in manual assertion
Supporting statements:
The phosphorylated form of the Sam68 N-terminus was still able to bind the G8.5 RNA but the intensity of the CSPs was strongly reduced compared with the non-phosphorylated regions (Figure 4C). This indicates that phosphorylation of T33 reduces but does not abolish its RNA binding ability. Analysis of the NMR titration experiment suggests that the Kd of the phosphorylated Sam68 N-terminus for the G8.5 RNA is ∼100 μM (Supplementary Figure S4), significantly higher than the Kd with the unphosphorylated form.
Together, our data demonstrate that the N- and C-terminal regions of Sam68 are capable of binding RNA and that phosphorylation of T33 and T317 by Cdk1 significantly reduces their RNA binding ability.
G8.5 is a RNA that contains the UAAA motif and was previously identified as a high affinity Sam68 binder by SELEX.
Protein: DP01705 (UniProt Q07666)
Region: residues 136	to 140
Annotation: molecular function inhibitor activity (Molecular function)
Evidence: heteronuclear single quantum coherence spectroscopy evidence used in manual assertion
Supporting statements:
Analysis of the NMR titration experiment suggests that the Kd of the phosphorylated Sam68 N-terminus for the G8.5 RNA is ∼100 μM (Supplementary Figure S4), significantly higher than the Kd with the unphosphorylated form. For the phosphorylated C-terminus, the chemical shift changes upon RNA addition were too small (Figure 4D) to allow us to derive a meaningful estimation of the Kd, suggesting that the dissociation constant of the T317-phosphoryled C-terminus to the RNA is >100 μM.
Together, our data demonstrate that the N- and C-terminal regions of Sam68 are capable of binding RNA and that phosphorylation of T33 and T317 by Cdk1 significantly reduces their RNA binding ability.
G8.5 is a RNA that contains the UAAA motif and was previously identified as a high affinity Sam68 binder by SELEX.
Protein: DP01705 (UniProt Q07666)
Region: residues 136	to 140
Annotation: regulation of localization (Biological process)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
As expected, WT Sam68 is mainly diffused in the nucleus with sometimes few SNBs (76.2 ± 6.3%, pattern A), with a small proportion of cells displaying Sam68 exclusively in SNBs (9.7 ± 5.7%, pattern B) or localized in both the nucleus and the cytoplasm (9.0 ± 4.6%, pattern C) (Figure 5B). In contrast, the T33E/T317E mutant displayed significant localization differences when compared with WT Sam68, with a significant decrease in the percentage of cells displaying pattern A (60.1 ± 4.9) and a 2-fold increase in the percentage of cells displaying pattern B (23.2 ± 4.5) (Figure 5B). These changes in localization are similar to previously reported mutations in the KH RNA-binding domain of Sam68 (51).
T33 and T317 mutations alter the cellular localization of Sam68
Consider T33E/T317E is a phospho-mimetic mutant.
Protein: DP01705 (UniProt Q07666)
Region: residues 31	to 35
Annotation: regulation of localization (Biological process)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
As expected, WT Sam68 is mainly diffused in the nucleus with sometimes few SNBs (76.2 ± 6.3%, pattern A), with a small proportion of cells displaying Sam68 exclusively in SNBs (9.7 ± 5.7%, pattern B) or localized in both the nucleus and the cytoplasm (9.0 ± 4.6%, pattern C) (Figure 5B). In contrast, the T33E/T317E mutant displayed significant localization differences when compared with WT Sam68, with a significant decrease in the percentage of cells displaying pattern A (60.1 ± 4.9) and a 2-fold increase in the percentage of cells displaying pattern B (23.2 ± 4.5) (Figure 5B). These changes in localization are similar to previously reported mutations in the KH RNA-binding domain of Sam68 (51).
T33 and T317 mutations alter the cellular localization of Sam68
Consider T33E/T317E is a phospho-mimetic mutant.
Protein: DP01705 (UniProt Q07666)
Region: residues 31	to 35
Annotation: regulation of RNA splicing (Biological process)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
As previously reported, transfection of WT Sam68 induced the inclusion of exon v5 in CD44 (Figure 6A) and increased the Bcl-xS/Bcl-xL ratio (Figure 6B). However, the T33E/T317E mutant was significantly less efficient at including CD44 exon v5 compared with the WT Sam68 (0.27 ± 0.01 for T33E/T317E compared with 0.31 ± 0.00 for WT Sam68), and both mutants were less efficient at shifting splicing towards the XS isoform [XS/(XL + XS) ratio of 0.06 ± 0.02 and 0.07 ± 0.01 for T33A/T317A and T33E/T317E compared with 0.09 ± 0.01 for WT Sam68]. Hence, phosphorylation of Sam68 T33 and T317 attenuates Sam68 regulatory activity on CD44 and Bcl-x splicing.
T33 and T317 mutants have reduced Sam68 splicing activity
Consider T33E/T317E as a phospho-mimetic mutant.
Protein: DP01705 (UniProt Q07666)
Region: residues 136	to 140
Annotation: regulation of RNA splicing (Biological process)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
As previously reported, transfection of WT Sam68 induced the inclusion of exon v5 in CD44 (Figure 6A) and increased the Bcl-xS/Bcl-xL ratio (Figure 6B). However, the T33E/T317E mutant was significantly less efficient at including CD44 exon v5 compared with the WT Sam68 (0.27 ± 0.01 for T33E/T317E compared with 0.31 ± 0.00 for WT Sam68), and both mutants were less efficient at shifting splicing towards the XS isoform [XS/(XL + XS) ratio of 0.06 ± 0.02 and 0.07 ± 0.01 for T33A/T317A and T33E/T317E compared with 0.09 ± 0.01 for WT Sam68]. Hence, phosphorylation of Sam68 T33 and T317 attenuates Sam68 regulatory activity on CD44 and Bcl-x splicing.
T33 and T317 mutants have reduced Sam68 splicing activity
Consider T33E/T317E as a phospho-mimetic mutant.
Protein: DP01705 (UniProt Q07666)
Region: residues 136	to 140
Annotation: regulation of cell cycle (Biological process)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
T33 and T317 mutants affect cell cycle progression, decrease apoptosis and increase proliferation
Expression of Sam68 WT significantly decreased (from 35.9 ± 3.7% to 26.0 ± 1.8%) the G2/M population and significantly increased (from 1.2 ± 0.1% to 16.2 ± 0.8%) the sub-G1 population compared with untransfected cells. In contrast, expression of Sam68 T33A/T317A and T33E/T317E mutants resulted in increased G2/M populations (30.4 ± 5.2% and 27.5 ± 1.2, respectively) compared with WT expression. Moreover, Sam68 T33E/T317E transfection resulted in a significant decrease in the sub-G1 population (14.0 ± 0.1%) and S population (15.3 ± 0.2), and an increase in the G1 population (43.2 ± 1.0%) compared with the WT.
Altogether, our data demonstrate that phosphorylation of Sam68 at T33 and T317 alters its regulatory role in cell cycle progression and apoptosis, leading to increased proliferation of HCT116 cells.
Consider T33E/T317E is a phospho-mimetic mutant.
Protein: DP01705 (UniProt Q07666)
Region: residues 31	to 35
Annotation: regulation of cell cycle (Biological process)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
T33 and T317 mutants affect cell cycle progression, decrease apoptosis and increase proliferation
Expression of Sam68 WT significantly decreased (from 35.9 ± 3.7% to 26.0 ± 1.8%) the G2/M population and significantly increased (from 1.2 ± 0.1% to 16.2 ± 0.8%) the sub-G1 population compared with untransfected cells. In contrast, expression of Sam68 T33A/T317A and T33E/T317E mutants resulted in increased G2/M populations (30.4 ± 5.2% and 27.5 ± 1.2, respectively) compared with WT expression. Moreover, Sam68 T33E/T317E transfection resulted in a significant decrease in the sub-G1 population (14.0 ± 0.1%) and S population (15.3 ± 0.2), and an increase in the G1 population (43.2 ± 1.0%) compared with the WT.
Altogether, our data demonstrate that phosphorylation of Sam68 at T33 and T317 alters its regulatory role in cell cycle progression and apoptosis, leading to increased proliferation of HCT116 cells.
Consider T33E/T317E is a phospho-mimetic mutant.
Protein: DP01705 (UniProt Q07666)
Region: residues 31	to 35
Annotation: regulation of apoptotic process (Biological process)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
T33 and T317 mutants affect cell cycle progression, decrease apoptosis and increase proliferation
Compared with untransfected cells, transfection of Sam68 WT increased the percentage of cells undergoing apoptosis as expected (from 12.1 ± 1.6% to 23.0 ± 1.7%), but this increase was significantly smaller upon transfection of the mutants (18.8 ± 1.5 and 16.6 ± 2.0 for T33AT317A and T33E/T317E, respectively) (Figure 7B).
Altogether, our data demonstrate that phosphorylation of Sam68 at T33 and T317 alters its regulatory role in cell cycle progression and apoptosis, leading to increased proliferation of HCT116 cells.
Consider T33E/T317E is a phospho-mimetic mutant.
Protein: DP01705 (UniProt Q07666)
Region: residues 136	to 140
Annotation: regulation of apoptotic process (Biological process)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
T33 and T317 mutants affect cell cycle progression, decrease apoptosis and increase proliferation
Compared with untransfected cells, transfection of Sam68 WT increased the percentage of cells undergoing apoptosis as expected (from 12.1 ± 1.6% to 23.0 ± 1.7%), but this increase was significantly smaller upon transfection of the mutants (18.8 ± 1.5 and 16.6 ± 2.0 for T33AT317A and T33E/T317E, respectively) (Figure 7B).
Altogether, our data demonstrate that phosphorylation of Sam68 at T33 and T317 alters its regulatory role in cell cycle progression and apoptosis, leading to increased proliferation of HCT116 cells.
Consider T33E/T317E is a phospho-mimetic mutant.
Protein: DP01714 (UniProt P0A8H8)
Region: residues 40	to 65
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The first 3 and last 25 residues are unstructured and shown in a random configuration.
Cross-references: PDB:1LV3
Protein: DP01716 (UniProt P0AD49)
Region: residues 91	to 113
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The 23 C-terminal residues of the protein are disordered. 
The protein has a βαβββα arrangement of secondary elements and a
disordered 23-residue C-terminal tail
The 23 C-terminal residues are intrinsically disordered as follows from
their relaxation properties
The 23 C-terminal residues show significantly lower H-15N heteronuclear NOE values and decreased R2, indicating that they are completely disordered in the solution.
Cross-references: PDB:1L4S
BMRB:5315
Protein: DP01720 (UniProt Q9NT62)
Region: residues 129	to 158
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Two of the Anchor region-containing IDRs for which binding partners have not yet been identified, ATG3 residues 129-158 and ATG9A residues 810-839, also do not undergo a disorder-to-helix transition in either 25% or 40 % TFE (Figure 2). These IDRs contain two or fewer helical residues (Table 5) even in 25% and 40% TFE, which is insufficient for the formation of a single stable helical turn
Protein: DP01724 (UniProt O95644)
Region: residues 391	to 400
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The absence of electron density for NFATc1LxVP residues 391–400 suggests that these residues remain flexible upon complex formation.
Cross-references: PDB:5SVE
Protein: DP01724 (UniProt O95644)
Region: residues 416	to 425
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Residues 1-12 and 173-178 are highly disordered.
The numbering above the sequence corresponds to that of NFATc-DBD, which corresponds to that of human NFATc (ref. 10) minus 413.
Cross-references: PDB:1NFA
Protein: DP01724 (UniProt O95644)
Region: residues 538	to 559
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
In NF-KB pSO, the 67 residues of the insert region form a compact helical bundle (Fig. 2b, red) which packs tightly against the immunoglobulin-like β-barrel2,3, whereas the much smaller insert region of NFATc-DBD (21 residues) projects out into solution and is unstructured.
Cross-references: PDB:1NFA
Protein: DP01724 (UniProt O95644)
Region: residues 384	to 398
Annotation: protein phosphatase 2B binding (Molecular function)
Evidence: glutathione S-transferase pull-down assay evidence used in manual assertion
Supporting statements:
Two polypeptide sequences, CnBP-A and another located near the CO2H-terminal end of the regulatory region, CnBP-B, were found to interact strongly with Cn (refer to Fig. 2b). Further truncations indicated the minimal CnBP-A and CnBP-B to comprise residues 118–131 and 384–398, respectively (data not shown)
Protein: DP01724 (UniProt O95644)
Region: residues 1	to 414
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The CD spectrum of unphosphorylated NFAT1–414 (Fig. 1b) closely resembles that of an unfolded protein (34) and specifically lacks the spectral features characteristic of α-helical or β-sheet secondary structure (Fig. 1b).
Protein: DP01724 (UniProt O95644)
Region: residues 1	to 414
Annotation: disorder (Structural state)
Evidence: cleavage assay evidence used in manual assertion
Supporting statements:
Consistent with the notion that unphosphorylated NFAT1–414 possesses little, if any, folded structure, the protein is degraded rapidly on exposure to 500 ng/ml of trypsin or chymotrypsin, whereas the folded core domain of Cn A remained intact under these conditions (data not shown).
Protein: DP01724 (UniProt O95644)
Region: residues 118	to 131
Annotation: protein phosphatase 2B binding (Molecular function)
Evidence: glutathione S-transferase pull-down assay evidence used in manual assertion
Supporting statements:
Two polypeptide sequences, CnBP-A and another located near the CO2H-terminal end of the regulatory region, CnBP-B, were found to interact strongly with Cn (refer to Fig. 2b). Further truncations indicated the minimal CnBP-A and CnBP-B to comprise residues 118–131 and 384–398, respectively (data not shown).
Protein: DP01724 (UniProt O95644)
Region: residues 384	to 398
Annotation: protein phosphatase 2B binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
These NMR results are consistent with those of truncation assays, indicating that the segment of CnBP-B most important for binding to Cn comprises residues 384–398.
Protein: DP01724 (UniProt O95644)
Region: residues 704	to 943
Annotation: disorder (Structural state)
Evidence: author inference based on intrinsic disorder prediction used in manual assertion
Supporting statements:
NFATs are of our particular interest because they have been confirmed by CD and NMR to contain extremely long disordered domains besides the welldefined DNA-binding domain[62]. These experimental data agree very well with the disorder prediction in Fig.6A.
PONDR FIT was used for all of the disorder predictions reported herein.
Figure 6A shows this region is predicted as disordered. The authors prediction also matches AlphaFold prediction and therefore we consider this region as disordered.
Protein: DP01729 (UniProt P69490)
Region: residues 144	to 159
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The structurally less-well-defined C-terminal subdomain contains a single helical turn (Figure 3B) followed by an unstructured tail of 16 residues (not shown in the structure).
Cross-references: PDB:1SR3
Protein: DP01730 (UniProt Q7Z3C6)
Region: residues 810	to 839
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Secondary structure content estimated by analysis of CD spectra for each of the thirteen IDRs showed that none contained more than two residues in a helical conformation (Figures 1-3, Table 3-5), which is insufficient for the formation of a stable α-helix requiring a minimum of six residues. Formation of a minimal beta-structure requires six residues [38], and seven of the thirteen IDRs (Table 3-5) appear to have between six to eight residues in extended or β-conformation which may be sufficient for forming short stable β-hairpins (Table 3-5). Thus, while none of the selected IDRs had stable helical structure, several may have short β-strands.
Protein: DP01730 (UniProt Q7Z3C6)
Region: residues 1	to 35
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Missing residue region in the cryo-EM structure of the full-length protein.
Cross-references: EMDB:EMD-21876
PDB:6WR4
Protein: DP01730 (UniProt Q7Z3C6)
Region: residues 96	to 105
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Missing residue region in the cryo-EM structure of the full-length protein.
Cross-references: EMDB:EMD-21876
PDB:6WR4
Protein: DP01730 (UniProt Q7Z3C6)
Region: residues 523	to 839
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Missing residue region in the cryo-EM structure of the full-length protein.
Cross-references: EMDB:EMD-21876
PDB:6WR4
Protein: DP01732 (UniProt Q06628)
Region: residues 269	to 738
Annotation: disorder (Structural state)
Evidence: atomic force microscopy evidence used in manual assertion
Supporting statements:
The Intrinsically Disordered Protein Atg13 Mediates Supramolecular Assembly of Autophagy Initiation Complexes
An essential autophagy factor Atg13 has a large intrinsically disordered region (IDR)
Using structural and biological techniques, we herein demonstrate that Atg13 has a large intrinsically disordered region (IDR) and interacts with two distinct Atg17 molecules using two binding regions in the IDR.
In the HS-AFM images of Atg13-MBP, the linkage of two globules by a string-like structure was apparent (Figure 1F and Movie S1). One of the globules was often observed to undergo transitions between ordered and disordered states within 1 s, whereas the other globule was always in an ordered state, indicating that the former is Atg13HORMA while the latter is MBP. The string-like structure is constantly disordered and highly flexible (Movie S1). This clearly demonstrates that the C-terminal region of Atg13 is actually intrinsically disordered, and thus is named IDR hereafter.
The IDR corresponds to the C-terminal region (residues 269–738).
Protein: DP01732 (UniProt Q06628)
Region: residues 268	to 738
Annotation: disorder (Structural state)
Evidence: atomic force microscopy evidence used in manual assertion
Supporting statements:
The IDR of Atg13 exhibited temporarily appearing and disappearing small globules, resulting in a double-Gaussian distribution of R_2D. Since the four small regions within the IDR are known to interact with Atg1 or Atg17, the small globules probably correspond to these regions.
Protein: DP01732 (UniProt Q06628)
Region: residues 424	to 436
Annotation: protein binding (Molecular function)
Evidence: qualitative western immunoblotting evidence used in manual assertion
Supporting statements:
We previously identified the first Atg17-binding region of Atg13, named Atg1317BR (residues 424–436), in which Ser428 and Ser429 are phosphorylated under nutrient-rich conditions but are dephosphorylated in response to starvation, thus enhancing the interaction with Atg17 (Fujioka et al., 2014). Immunoprecipitation analysis showed that Atg17 efficiently co-precipitated with GFP-Atg13 (Figure 1A, lanes 11 and 12), but not with GFP-Atg13 variants containing the S429D or F430A mutation in Atg1317BR (Figure 1A, lanes 13–16), confirming that Atg1317BR is crucial for the interaction with Atg17.
The IDR of Atg13 contains two distinct regions binding intermolecularly to Atg17
We also revealed that Atg13 interacts with Atg17 via a small portion (residues 424–436), called the Atg17-binding region (Atg1317BR), and found that the Atg1317BR-Atg17 interaction is also promoted by starvation-dependent dephosphorylation of Ser428 and Ser429 in Atg1317BR.
Protein: DP01732 (UniProt Q06628)
Region: residues 359	to 389
Annotation: protein binding (Molecular function)
Evidence: isothermal titration calorimetry evidence used in manual assertion
Supporting statements:
We next investigated the binding affinity of the Atg1317LR-Atg17 interaction by ITC. This analysis showed that a synthetic Atg1317LR peptide (residues 359–389) bound to Atg17 with a KD value of 2.0 μM (Figure 1E), indicating that although the Atg1317LR-Atg17 interaction is ∼10-fold weaker than the Atg1317BR-Atg17 interaction (Figure 1B, 220 nM), Atg1317LR can interact with Atg17 independently of Atg1317BR.
The IDR of Atg13 contains two distinct regions binding intermolecularly to Atg17
Protein: DP01732 (UniProt Q06628)
Region: residues 359	to 389
Annotation: PAS complex (Cellular component)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
Because Atg13 is a key factor that tethers Atg1 and Atg17 to form the Atg1 complex (Fujioka et al., 2014), we next investigated whether the Atg1317LR-Atg17 interaction is required for PAS organization. Fluorescence microscopy revealed that in cells expressing wild-type Atg13 or the S379A mutant, Atg17-GFP assembled to form the PAS upon rapamycin treatment (Figure 3D). However, in cells expressing the F375A mutant, the PAS was not observed at all (Figure 3D), and the S379D mutant was also defective in the PAS formation (Figure 3D). These observations indicate that the Atg1317LR-Atg17 interaction is required for PAS assembly.
Atg1317LR-Atg17 Interaction Is Crucial for PAS Assembly and Autophagy In Vivo