LiteFold/DisProt huggingface dataset:
Protein: DP01732 (UniProt Q06628)
Region: residues 424	to 436
Annotation: Atg1/ULK1 kinase complex (Cellular component)
Evidence: analytical ultracentrifugation evidence used in manual assertion
Supporting statements:
We next tested the intermolecular binding model (Figure 4E, right). For this purpose, we examined whether a short fragment of Atg13 containing Atg1317LR and Atg1317BR (residues 359–459) can interlink the Atg17-Atg29-Atg31 complexes in vitro (Figure 5A). By centrifugation, the Atg17-Atg29-Atg31 complex alone was almost completely recovered in the supernatant fraction (Figure 5A, lane 1). However, on addition of the Atg13 fragment, nearly half of the Atg17-Atg29-Atg31 complex became precipitable (Figure 5A, lane 4), probably due to aggregate formation by excess interlinking among the Atg13 fragments and the Atg17-Atg29-Atg31 complexes. In contrast, the addition of an Atg13 fragment bearing a mutation in Atg1317LR (S379D) or Atg1317BR (S429D) did not result in the precipitation of the Atg17-Atg29-Atg31 complex by centrifugation (Figure 5A, lanes 6 and 8).
These results suggest that the short Atg13 fragment can link the Atg17-Atg29-Atg31 complexes with each other via Atg1317LR and Atg1317BR, irrespective of other Atg13 regions such as Atg1317BR2. These results strongly support the intermolecular binding of Atg1317LR and Atg1317BR to the Atg17-Atg29-Atg31 complexes.
We further investigated the formation of the large Atg1 complexes by analytical ultracentrifugation. By use of wild-type Atg13-MBP, we observed large assemblages with sedimentation coefficient(s) of 15–20 S (Figure 5B). By contrast, when we used the Atg1317LR and Atg1317BR mutant, the 15- to 20-S supramolecular complex was barely detectable; instead, we observed a moderately sized complex with sedimentation coefficient of ∼10 S (Figure 5B), still larger than the Atg17-Atg29-Atg31 complex alone (sedimentation coefficient of ∼5 S). These results suggest that Atg13 mediates multimeric assembly of the Atg1 complexes via Atg1317LR and Atg1317BR, leading in turn to generation of the 15- to 20-S supramolecular complexes.
Protein: DP01732 (UniProt Q06628)
Region: residues 359	to 389
Annotation: Atg1/ULK1 kinase complex (Cellular component)
Evidence: analytical ultracentrifugation evidence used in manual assertion
Supporting statements:
We next tested the intermolecular binding model (Figure 4E, right). For this purpose, we examined whether a short fragment of Atg13 containing Atg1317LR and Atg1317BR (residues 359–459) can interlink the Atg17-Atg29-Atg31 complexes in vitro (Figure 5A). By centrifugation, the Atg17-Atg29-Atg31 complex alone was almost completely recovered in the supernatant fraction (Figure 5A, lane 1). However, on addition of the Atg13 fragment, nearly half of the Atg17-Atg29-Atg31 complex became precipitable (Figure 5A, lane 4), probably due to aggregate formation by excess interlinking among the Atg13 fragments and the Atg17-Atg29-Atg31 complexes. In contrast, the addition of an Atg13 fragment bearing a mutation in Atg1317LR (S379D) or Atg1317BR (S429D) did not result in the precipitation of the Atg17-Atg29-Atg31 complex by centrifugation (Figure 5A, lanes 6 and 8).
These results suggest that the short Atg13 fragment can link the Atg17-Atg29-Atg31 complexes with each other via Atg1317LR and Atg1317BR, irrespective of other Atg13 regions such as Atg1317BR2. These results strongly support the intermolecular binding of Atg1317LR and Atg1317BR to the Atg17-Atg29-Atg31 complexes.
We further investigated the formation of the large Atg1 complexes by analytical ultracentrifugation. By use of wild-type Atg13-MBP, we observed large assemblages with sedimentation coefficient(s) of 15–20 S (Figure 5B). By contrast, when we used the Atg1317LR and Atg1317BR mutant, the 15- to 20-S supramolecular complex was barely detectable; instead, we observed a moderately sized complex with sedimentation coefficient of ∼10 S (Figure 5B), still larger than the Atg17-Atg29-Atg31 complex alone (sedimentation coefficient of ∼5 S). These results suggest that Atg13 mediates multimeric assembly of the Atg1 complexes via Atg1317LR and Atg1317BR, leading in turn to generation of the 15- to 20-S supramolecular complexes.
Protein: DP01732 (UniProt Q06628)
Region: residues 424	to 436
Annotation: Atg1/ULK1 kinase complex (Cellular component)
Evidence: chromatography evidence used in manual assertion
Supporting statements:
Gel-filtration analysis showed that recombinant Atg1ΔKD-Atg13-MBP and Atg17-Atg29-Atg31 subcomplexes were distributed in fractions 15–16 and 12–14, respectively (Figures 5C and S5A). Upon mixing these two subcomplexes (10 μM each), all five components were co-distributed in large fractions (fractions 5–8) in a manner dependent on both Atg1317BR and Atg1317LR (Figure S5A). These results suggest that the five components interact with each other to form large complexes, a process in which Atg1317LR and Atg1317BR play an essential role.
Protein: DP01732 (UniProt Q06628)
Region: residues 359	to 389
Annotation: Atg1/ULK1 kinase complex (Cellular component)
Evidence: chromatography evidence used in manual assertion
Supporting statements:
Gel-filtration analysis showed that recombinant Atg1ΔKD-Atg13-MBP and Atg17-Atg29-Atg31 subcomplexes were distributed in fractions 15–16 and 12–14, respectively (Figures 5C and S5A). Upon mixing these two subcomplexes (10 μM each), all five components were co-distributed in large fractions (fractions 5–8) in a manner dependent on both Atg1317BR and Atg1317LR (Figure S5A). These results suggest that the five components interact with each other to form large complexes, a process in which Atg1317LR and Atg1317BR play an essential role.
Protein: DP01732 (UniProt Q06628)
Region: residues 424	to 436
Annotation: positive regulation of autophagy (Biological process)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
We next studied the importance of the Atg13-mediated self-assembly of Atg1 complexes in vivo. Consistent with the in vitro biochemical analyses, fluorescence microscopy showed that PAS assembly of Atg17-GFP and Atg13-mCherry (i.e., the multimeric assembly of the Atg1 complexes in vivo) strictly required both Atg1317BR and Atg1317LR (Figure 6A): upon mutation in Atg1317BR or Atg1317LR, the PAS was barely detectable (Figure 6A), even when PAS formation was enhanced using a kinase-dead form of Atg1 (D211A) (Sekito et al., 2009).
Altogether, these data indicate that Atg13 links the Atg17-Atg29-Atg31 complexes via two distinct regions, Atg1317BR and Atg1317LR, thus leading to multimeric assembly of Atg1 complexes.
Atg13-Mediated Self-Assembly of Atg1 Complexes Is Required for Initial Events in Autophagy
Protein: DP01732 (UniProt Q06628)
Region: residues 571	to 700
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Atg13[571-700] is intrinsically disordered
After screening several C-terminal constructs for expression and solubility in E. coli, we succeeded in purifying Atg13[571–700]. As expected, this region was intrinsically disordered as observed by 2D 1H-15N HSQC spectroscopy and circular dichroism (CD) (Figures S1 and S2).
Protein: DP01732 (UniProt Q06628)
Region: residues 571	to 700
Annotation: lipid binding (Molecular function)
Evidence: co-sedimentation assay evidence used in manual assertion
Supporting statements:
Atg13[571-700] binds negatively charged liposomes
Full-length Atg13 was shown to bind yeast polar lipids [2], suggesting that electrostatic interactions may play an important role in liposome binding by the Atg13[571–700] peptide. To test if this is the case, we performed liposome sedimentation assays in the presence of increasing concentrations of NaCl (Figure 1D,E). If electrostatic interactions are required for membrane binding, then increasing NaCl concentrations will disrupt this interaction. We found that Atg13[571–700] bound to Folch liposomes in buffer containing either 50 or 150 mM NaCl, but that binding was almost completely abolished in buffer containing 300 or 500 mM NaCl. This result suggests that Atg13 binding to liposomes is mediated primarily through electrostatic interactions. In agreement with this result, Atg13[571–700] also bound yeast polar lipids, and this binding was severely disrupted by a NaCl concentration higher than 50 mM (Figure 1 F,G).
Protein: DP01732 (UniProt Q06628)
Region: residues 639	to 643
Annotation: lipid binding (Molecular function)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
To further examine the role of the 640KFK and 683KFHK motifs in liposome binding, we mutated the Atg13[571–700] sequence to F641G I645G, F641E I645E, or K683E H685E K686E. Liposome sedimentation assays were performed in 50, 150, 300 and 500 mM NaCl. All 3 mutants were significantly defective in membrane binding at a physiological concentration of 150 mM NaCl, with more than 60% reduction relative to wild type (Figure 2B). A summary of all truncation and mutagenesis data is presented in Figure S5. These data suggest that Atg13[571–700] contains 2 distinct liposome-binding motifs, 640KFK (site I) and 683KFHK (site II) (Figure 1A) and that electrostatic force is not a sole factor responsible for lipid binding of the Atg13[571–700] region. A hydrophobic insertion of aromatic residues in the motifs plays a role in binding efficiency, in agreement with data in Figure 1.
Region including the first charged motif, KFK.
Protein: DP01732 (UniProt Q06628)
Region: residues 682	to 687
Annotation: lipid binding (Molecular function)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
To further examine the role of the 640KFK and 683KFHK motifs in liposome binding, we mutated the Atg13[571–700] sequence to F641G I645G, F641E I645E, or K683E H685E K686E. Liposome sedimentation assays were performed in 50, 150, 300 and 500 mM NaCl. All 3 mutants were significantly defective in membrane binding at a physiological concentration of 150 mM NaCl, with more than 60% reduction relative to wild type (Figure 2B). A summary of all truncation and mutagenesis data is presented in Figure S5. These data suggest that Atg13[571–700] contains 2 distinct liposome-binding motifs, 640KFK (site I) and 683KFHK (site II) (Figure 1A) and that electrostatic force is not a sole factor responsible for lipid binding of the Atg13[571–700] region. A hydrophobic insertion of aromatic residues in the motifs plays a role in binding efficiency, in agreement with data in Figure 1.
Region including the second charged motif, KFHK.
Protein: DP01732 (UniProt Q06628)
Region: residues 660	to 686
Annotation: protein binding (Molecular function)
Evidence: isothermal titration calorimetry evidence used in manual assertion
Supporting statements:
To investigate whether mutations in the phospholipid-binding sites I and II of Atg13 (640KFK and 683KFHK) affect binding with Vac8, we tested the binding affinity of the Atg13 glycine and glutamate mutants with Vac8 relative to wild type. As indicated above, we also constructed a mutant with a presumably disrupted interaction between the Atg13 peptide and Vac8. The D651R Q666R P667R mutant carries cationic residues in the Vac8-binding region to repulse the peptide from the positively charged Vac8 groove. We applied isothermal calorimetry (ITC) to measure dissociation constants for Atg13[571–700] binding to recombinant purified Vac8[10–515]. The WT and all of the mutated Atg13[571–700] peptides, except for Atg13[571–700]D651,Q666,P667R, bound to Vac8 with dissociation constants from 227–736 nM (Table 1), confirming that the Atg13[571–700] region carries the Vac8-binding domain, and that the Atg13 residues required for liposome binding are not required for Vac8 binding (Figure 3A). Atg13[571–700]D651,Q666,P667R bound to Vac8 with the lowest affinity (Kd of 1885 nM; Table 1), demonstrating that the Atg13 region between the phospholipid site I and II (Figure S6A) is involved in Vac8 binding, in agreement with the crystallographic data.
Protein: DP01732 (UniProt Q06628)
Region: residues 571	to 700
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The 2D 1 H-15 N HSQC spectra of Atg13[571-700]. The lack of dispersion in the amide region of the spectrum is indicative that this region is intrinsically disordered.
Protein: DP01732 (UniProt Q06628)
Region: residues 571	to 700
Annotation: positive regulation of autophagy (Biological process)
Evidence: phosphatase assay evidence used in manual assertion
Supporting statements:
The lipid- and Vac8-binding capability of Atg13 are required for efficient autophagy
To determine if nonselective autophagy activity is affected by a chromosomal mutation in the phospholipid- and Vac8-binding motifs of the full-length Atg13 protein, we performed the quantitative Pho8Δ60 assay, in which Pho8Δ60 is a cytosolic zymogen of the vacuolar Pho8 phosphatase that lacks the first 60 amino acids of its N terminus. Vacuolar localization and activation of this zymogen relies on nonselective autophagy [22]. Cells with wild-type (WT) Atg13 exhibited a substantial increase in Pho8Δ60 activity after 3 h of nitrogen starvation (Figure 5A). In comparison, the F641E I645E, K683E H685E K686E, and D651R Q666R P667R mutants showed an ~30% decrease in Pho8Δ60 activity; the F641G I645G mutant was even more defective, as it exhibited an ~50% decrease in autophagy activity under the same experimental conditions.
Atg13 IDR mutants reduce autophagy activity. (A) Autophagy activity was measured by the Pho8Δ60 assay in WT and atg13∆ strains, and in cells expressing the F641E I645E, F641G I645G, K683E H685E K686E, or D651R Q666R P667R mutants under nutrient-rich conditions (+N) and after 3 h of nitrogen starvation (-N 3 h).
Autophagy was measured using the GFP-Atg8 processing assay in the same strains as in (A) under nutrient-rich conditions and after 1 or 2 h of nitrogen starvation.
The summary diagram of the lipid- and Vac8-binding capability and autophagy activity of the Atg13 mutants (Figure 5D) shows that the Atg13 protein promotes efficient autophagy only when its interaction with both phospholipids and Vac8 is intact and thereby balanced.
Protein: DP01732 (UniProt Q06628)
Region: residues 571	to 700
Annotation: regulation of cellular localization (Biological process)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
Again, these data suggest that the Atg13-phospholipd interaction needs to be effective and in concert with the Atg13-Vac8 interaction to maintain an optimal balance in localization of Atg13 in proximity to the vacuole; too little or too much binding to either component is detrimental to autophagy (Figures 5 and 6).
Protein: DP01732 (UniProt Q06628)
Region: residues 359	to 389
Annotation: positive regulation of autophagy (Biological process)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
Atg1317LR is essential for autophagy.
The Atg1317LR-binding region of Atg17 is required for autophagy.
We further examined several Atg17 mutants by the ALP assay and found that the I385A, P393A, and T395A mutants exhibited severe defects in autophagy (Figure 3F).
Furthermore, Atg1317LR and Atg1317BR were strictly required for autophagy as estimated by the ALP assay (Figure 4C). However, deletion of or mutations in Atg1317BR2 only slightly reduced autophagic activity even in the atg11Δ background (Figure 4C). We also found that GFP-Atg13 bearing a double mutation in Atg1317LR and Atg1317BR (F375A F430A) did not precipitate Atg17 at all (Figure 4D, lane 16), even though it still contains Atg1317BR2. It should be noted that to detect weak interactions between Atg13 and Atg17, we used a mild detergent condition (0.1% NP-40) in this experiment. These results indicate that Atg1317LR and Atg1317BR, but not Atg1317BR2, are responsible for starvation-induced autophagy.
Region corresponding to Atg1317LR peptide (residues 359–389).
Protein: DP01732 (UniProt Q06628)
Region: residues 424	to 436
Annotation: positive regulation of autophagy (Biological process)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
Atg1317LR is essential for autophagy.
The Atg1317LR-binding region of Atg17 is required for autophagy.
We further examined several Atg17 mutants by the ALP assay and found that the I385A, P393A, and T395A mutants exhibited severe defects in autophagy (Figure 3F).
Furthermore, Atg1317LR and Atg1317BR were strictly required for autophagy as estimated by the ALP assay (Figure 4C). However, deletion of or mutations in Atg1317BR2 only slightly reduced autophagic activity even in the atg11Δ background (Figure 4C). We also found that GFP-Atg13 bearing a double mutation in Atg1317LR and Atg1317BR (F375A F430A) did not precipitate Atg17 at all (Figure 4D, lane 16), even though it still contains Atg1317BR2. It should be noted that to detect weak interactions between Atg13 and Atg17, we used a mild detergent condition (0.1% NP-40) in this experiment. These results indicate that Atg1317LR and Atg1317BR, but not Atg1317BR2, are responsible for starvation-induced autophagy.
Region corresponding to Atg1317BR peptide (residues 424-436).
Protein: DP01732 (UniProt Q06628)
Region: residues 460	to 521
Annotation: disorder (Structural state)
Evidence: circular dichroism evidence used in manual assertion
Supporting statements:
CD spectra showed that Atg13 (460–521) is intrinsically disordered in solution (Supplementary Fig. 1c).
Protein: DP01732 (UniProt Q06628)
Region: residues 460	to 521
Annotation: protein binding (Molecular function)
Evidence: glutathione S-transferase pull-down assay evidence used in manual assertion
Supporting statements:
We found that 62 amino acids (residues 460–521) of Atg13 are necessary and sufficient for Atg1 binding (Supplementary Fig. 1b).
Protein: DP01733 (UniProt Q8AVN9)
Region: residues 1	to 12
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The N terminus from A2 to D12 and the linker between the two zinc-binding domains, from N69 to D92, are disordered. This is demonstrated by heteronuclear 1H–15N NOE measurements (Figure 4).
Cross-references: PDB:1ZU1
BMRB:6655
Protein: DP01733 (UniProt Q8AVN9)
Region: residues 69	to 92
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The N terminus from A2 to D12 and the linker between the two zinc-binding domains, from N69 to D92, are disordered. This is demonstrated by heteronuclear 1H–15N NOE measurements (Figure 4).
Cross-references: PDB:1ZU1
BMRB:6655
Protein: DP01733 (UniProt Q8AVN9)
Region: residues 69	to 92
Annotation: flexible linker (Disorder function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The N terminus from A2 to D12 and the linker between the two zinc-binding domains, from N69 to D92, are disordered. This is demonstrated by heteronuclear 1H–15N NOE measurements (Figure 4).
 We have determined the solution structure of a 127 residue fragment of ZFa, which consists of two zinc finger domains connected by a linker that remains unstructured in the free protein in solution.
Cross-references: BMRB:6655
PDB:1ZU1
Protein: DP01735 (UniProt P80644)
Region: residues 173	to 191
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The remaining 18 or 19 residues to the C-terminus (residue 191) had no clear electron density and were not modeled.
Cross-references: PDB:4PTY
Protein: DP01736 (UniProt P52597)
Region: residues 1	to 10
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Except for the 10 first residues that are highly flexible [negative (1H-15N)-NOE values], the 2 qRRMs possess a rigid core [(1H-15N)-NOE values higher than 0.7]. The linker region (residues 100–110), however, shows low (1H-15N)-NOE values (between 0.19 and 0.5), which indicates that the linker between the two qRRMs is flexible and suggests that the two qRRMs tumble independently in solution.
Cross-references: PDB:2HGL
PDB:2HGM
Protein: DP01736 (UniProt P52597)
Region: residues 100	to 110
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Except for the 10 first residues that are highly flexible [negative (1H-15N)-NOE values], the 2 qRRMs possess a rigid core [(1H-15N)-NOE values higher than 0.7]. The linker region (residues 100–110), however, shows low (1H-15N)-NOE values (between 0.19 and 0.5), which indicates that the linker between the two qRRMs is flexible and suggests that the two qRRMs tumble independently in solution.
Cross-references: PDB:2HGL
PDB:2HGM
Protein: DP01736 (UniProt P52597)
Region: residues 100	to 110
Annotation: flexible linker (Disorder function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Except for the 10 first residues that are highly flexible [negative (1H-15N)-NOE values], the 2 qRRMs possess a rigid core [(1H-15N)-NOE values higher than 0.7]. The linker region (residues 100–110), however, shows low (1H-15N)-NOE values (between 0.19 and 0.5), which indicates that the linker between the two qRRMs is flexible and suggests that the two qRRMs tumble independently in solution.
Cross-references: PDB:2HGL
PDB:2HGM
Protein: DP01737 (UniProt P9WKX3)
Region: residues 1	to 35
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
the characteristic circular dichroism (CD) signature observed for full-length Bpa, which features two minima at 208 and 220 nm (Figure 1B). Based on our in silico analysis, we generated a truncation variant (HisBpa36–159), from which the non-homologous and likely disordered regions were excluded. The CD spectrum of this variant shows the same overall shape with a slightly stronger helical signal
Protein: DP01737 (UniProt P9WKX3)
Region: residues 160	to 174
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
the characteristic circular dichroism (CD) signature observed for full-length Bpa, which features two minima at 208 and 220 nm (Figure 1B). Based on our in silico analysis, we generated a truncation variant (HisBpa36–159), from which the non-homologous and likely disordered regions were excluded. The CD spectrum of this variant shows the same overall shape with a slightly stronger helical signal
Protein: DP01738 (UniProt P04271)
Region: residues 18	to 28
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Thus, the poor definition of the calcium-binding loops in human apo-S100B is most consistent with flexibility of the backbone within these regions.
Cross-references: PDB:2PRU
Protein: DP01738 (UniProt P04271)
Region: residues 18	to 28
Annotation: calcium ion binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Calcium binding loop I
Protein: DP01738 (UniProt P04271)
Region: residues 80	to 91
Annotation: disorder (Structural state)
Evidence: proton-based nuclear magnetic resonance evidence used in manual assertion
Supporting statements:
In the tertiary structure of S100B, residues with exchange contributions are grouped around the mobile C-terminal loop
(Figure 4, panel C). Exchange is detected for all HSQC correlations from the last turn of helix 4 (V80) to the
C-terminus at E91, and no correlations are observed for residues H85, E86, and F87 due to exchange broadening. ..this region of the protein is mobile on the chemical shift time scale.