LiteFold/DisProt huggingface dataset:
Protein: DP02547 (UniProt Q9Y6G9)
Region: residues 388	to 523
Annotation: protein binding (Molecular function)
Evidence: microscale thermophoresis evidence used in manual assertion
Supporting statements:
the light intermediate chain (LIC) subunit of human dynein uses a short helix in its disordered C-terminal region to bind structurally distinct adaptor proteins that connect the motor to specific cargo.
Adaptors known to bind LIC1-C include BICD2 and Spindly (SPDL1), which are likely related [15,16], as well as the structurally distinct adaptors HOOK3, RAB11 family-interacting protein 3 (RAB11FIP3; hereafter referred to as FIP3), and RAB-interacting lysosomal protein (RILP) [12,17].
We also measured binding to fluorescently labeled LIC1(388–523) in microscale thermophoresis (MST) experiments. We obtained KD values of 13.1 ± 0.4 μM and 6.0 ± 1.3 μM for the interaction with SPDL1(2–359) and BICD2(2–422), respectively, which is in reasonable agreement with the SPR analysis
Protein: DP02547 (UniProt Q9Y6G9)
Region: residues 388	to 523
Annotation: protein binding (Molecular function)
Evidence: bait-prey hybrid interaction evidence used in manual assertion
Supporting statements:
the light intermediate chain (LIC) subunit of human dynein uses a short helix in its disordered C-terminal region to bind structurally distinct adaptor proteins that connect the motor to specific cargo.
Adaptors known to bind LIC1-C include BICD2 and Spindly (SPDL1), which are likely related [15,16], as well as the structurally distinct adaptors HOOK3, RAB11 family-interacting protein 3 (RAB11FIP3; hereafter referred to as FIP3), and RAB-interacting lysosomal protein (RILP) [12,17].
To directly test whether helix 1 is important for adaptor binding, we performed in vitro pull-down experiments using purified glutathione S-transferase (GST)-tagged versions of LIC1-C (Fig 3A and 3B) and LIC2-C (Fig 4A and 4B) as bait. In addition to BICD2(2–422), SPDL1(2–359), and HOOK3(2–552), we purified full-length versions of RILP (residues 1–401) and FIP3 (residues 2–756), as well as an N-terminal fragment of NIN (residues 1–693) (Fig 3C, S1B Fig). Using Coomassie Blue staining and immunoblotting for the Strep-tag II at the adaptor C terminus, all adaptors were readily detected in GST pull-downs with LIC1(388–523) and LIC1(388–471) (Fig 3D).
Protein: DP02548 (UniProt P0AES9)
Region: residues 22	to 110
Annotation: order to disorder (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
HdeA exists as a well-folded homodimer in the inactive state, and its activation requires acid-induced protein unfolding accompanied by dimer dissociation, resulting in the exposure of hydrophobic surfaces for interaction with denatured client proteins (9-10, 13, 18-23).
Protein: DP02548 (UniProt P0AES9)
Region: residues 22	to 110
Annotation: protein folding chaperone (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
HdeA exists as a well-folded homodimer in the inactive state, and its activation requires acid-induced protein unfolding accompanied by dimer dissociation, resulting in the exposure of hydrophobic surfaces for interaction with denatured client proteins (9-10, 13, 18-23).
Taking all the above factors into consideration, it is clear that the two segments that are rich in hydrophobic residues, namely the 28–35 and 49–55 segments, play central roles in directly binding to unfolded client proteins under acid stress.
Protein: DP02548 (UniProt P0AES9)
Region: residues 22	to 110
Annotation: protein folding chaperone (Molecular function)
Evidence: experimental phenotypic evidence used in manual assertion
Supporting statements:
hdeA gene deletion abolishes E. coli acid resistance in vivo
Protein: DP02548 (UniProt P0AES9)
Region: residues 22	to 110
Annotation: molten globule (Structural state)
Evidence: fluorescence resonance energy transfer evidence used in manual assertion
Supporting statements:
We show by intramolecular FRET that the partially unfolded character of active HdeA allows the chaperone to adopt different conformations as required for the recognition and high-affinity binding of different substrate proteins.
Protein: DP02548 (UniProt P0AES9)
Region: residues 22	to 110
Annotation: protein folding chaperone (Molecular function)
Evidence: combinatorial experimental and author inference evidence contained in single publication used in manual assertion
Supporting statements:
Our results are consistent with a model in which HdeA stably binds substrates at low pH, thereby preventing their irreversible aggregation. pH neutralization subsequently triggers the slow release of substrate proteins from HdeA, keeping the concentration of aggregation-sensitive intermediates below the threshold where they begin to aggregate. This provides a straightforward and ATP-independent mechanism that allows HdeA to facilitate protein refolding.
Protein: DP02548 (UniProt P0AES9)
Region: residues 22	to 110
Annotation: molten globule (Structural state)
Evidence: nuclear magnetic resonance spectroscopy-based hydrogen-deuterium exchange evidence used in manual assertion
Supporting statements:
The results indicate that, as the pH transitions to 4.0 and lower (and progressively falls below the pKas for aspartate and glutamate residues), the protein samples a larger range of conformations as a precursor to unfolding and transitioning to the monomer. Based on these results it appears that the dimer interface is one of the first segments within the HdeA dimer to start opening up, as shown by the large loss of solvent protection for helix B at pH 4.0.
Protein: DP02548 (UniProt P0AES9)
Region: residues 22	to 110
Annotation: protein binding (Molecular function)
Evidence: combinatorial experimental and author inference evidence contained in single publication used in manual assertion
Supporting statements:
"Our results are consistent with a model in which HdeA stably binds substrates at low pH, thereby preventing their irreversible aggregation. pH neutralization subsequently triggers the slow release of substrate proteins from HdeA, keeping the concentration of aggregation-sensitive intermediates below the threshold where they begin to aggregate. This provides a straightforward and ATP-independent mechanism that allows HdeA to facilitate protein refolding."
Protein: DP02548 (UniProt P0AES9)
Region: residues 22	to 110
Annotation: protein binding (Molecular function)
Evidence: cell aggregation evidence used in manual assertion
Supporting statements:
"A protein aggregation assay was carried out at pH 2 with HDEA (see Materials and Methods). The results illustrated in Figure 1(b) document that HDEA, at the equivalent mass concentration, prevents aggregation of denatured rhodanese under extremely acidic conditions in vitro."
Protein: DP02548 (UniProt P0AES9)
Region: residues 22	to 110
Annotation: protein folding chaperone (Molecular function)
Evidence: cell aggregation evidence used in manual assertion
Supporting statements:
"A protein aggregation assay was carried out at pH 2 with HDEA (see Materials and Methods). The results illustrated in Figure 1(b) document that HDEA, at the equivalent mass concentration, prevents aggregation of denatured rhodanese under extremely acidic conditions in vitro."
Protein: DP02549 (UniProt O82775)
Region: residues 1	to 180
Annotation: disorder (Structural state)
Evidence: sodium dodecyl sulfate polyacrylamide gel electrophoresis evidence used in manual assertion
Supporting statements:
We noticed that the migration rates of ATI1-N and ATI2-N in SDS-PAGE were slower than
expected by their molecular weights (MwATI1 -N = 20.46 kDa; MwATI2-N = 21.02 kDa) (Fig. 3A). This simple observation represents a first indication that ATI1 -N and ATI2-N may be intrinsically disordered, because IDRs are typically depleted in hydrophobic residues, and, consequently, tend to bind less SDS, explaining their abnormally slow mobility in SDS-PAGE [37].
Protein: DP02549 (UniProt O82775)
Region: residues 1	to 180
Annotation: disorder (Structural state)
Evidence: temperature-induced protein unfolding evidence used in manual assertion
Supporting statements:
Since we cannot exclude that ATI1-N and ATI2-N form folded dimers, we subjected ATI1-N,
ATI2-N and the globular GST to heat treatments for 10 min at 75°C or 5 min at 95°C, and separated
aggregated from soluble protein by centrifugation (Fig. 3C-D). These aggregation tests showed that,
in contrast to the GST control, ATI1-N and ATI2-N remained soluble upon subjection to elevated
temperatures that unfold most globular proteins from non-thermophilic organisms.
Protein: DP02549 (UniProt O82775)
Region: residues 1	to 180
Annotation: disorder (Structural state)
Evidence: chromatography evidence used in manual assertion
Supporting statements:
We next subjected protein samples of ATI1-N, ATI2-N and the similarly sized, globular prolactin (PRL, 23 kDa) to size-exclusion chromatography in which the elution volume decreases with the Stokes radius of the protein, such that folded proteins elute at larger volumes than IDPs of similar molecular mass. ATI2-N eluted at the smallest volume (67 ml), followed by ATI1-N (75 ml), and finally the globular PRL (85 ml) (Figure 3B), suggesting that the Stokes radii (r) increase in the order r(PRL) < r(ATI1-N) < r(ATI2-N).
Protein: DP02549 (UniProt O82775)
Region: residues 1	to 180
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The CD spectra of both ATI1-N and ATI2-N showed characteristic negative minima ∼200 nm, indicating a low content of secondary structure (Figure 4A). In addition, the broad minima at ∼220 nm may indicate some low (<10%) α-helical content [42], in agreement with the secondary structure prediction obtained utilizing the Psipred tool (Figure 4B) [43,44].
Protein: DP02549 (UniProt O82775)
Region: residues 1	to 180
Annotation: pre-molten globule (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
For ATI1-N, the results are less clear. Although most amide and methyl proton signals collapse, characteristic of IDPs and different from fully folded regions, a broader dispersion of signals both in the methyl region and in the amide region was observed compared to ATI2-N (Fig. 5).
The latter is often observed in disordered regions with transient structure formation and/or transient long-range contacts, consistent with the more compact nature of ATI1-N compared to ATI2-N as suggested by the size exclusion chromatography results.
Protein: DP02549 (UniProt O82775)
Region: residues 14	to 17
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
We next used the well-resolved indole NH peaks in the 1D 1H NMR spectra to query whether the ATI1-N and ATI2-N were sufficient for ATG8 interaction. First, we expressed wild type and W14A/V17A mutants of ATI1-N and ATI2-N (Figure 6B), and recorded 1D 1H NMR spectra to identify the peak corresponding to the indole NH of W14 in ATI1-N and ATI2-N (Figure 6C). We then recorded spectra in the presence and absence of purified ATG8f expressed in E. coli (Figure 6D,E), and examined the regions of the spectra with chemical shifts from the indole NH protons. This analysis showed clear line broadening of the W14 indole NH signal in the presence of ATG8f (Figure 6E), indicating that binding involves the N-terminal AIM of both ATI1-N and ATI2-N. We conclude that the N-terminal AIMs of ATI1 and ATI2 are necessary and sufficient for binding to ATG8f. We also conclude from the sharp peaks of the indole NH protons in the unbound state that the functional AIMs in both ATI1 and ATI2 reside in regions of high flexibility.
Region including the N-terminal AIM motif (WEVV) at position 14-17.
Protein: DP02549 (UniProt O82775)
Region: residues 14	to 17
Annotation: selective autophagy (Biological process)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
We next used the well-resolved indole NH peaks in the 1D 1H NMR spectra to query whether the ATI1-N and ATI2-N were sufficient for ATG8 interaction. First, we expressed wild type and W14A/V17A mutants of ATI1-N and ATI2-N (Figure 6B), and recorded 1D 1H NMR spectra to identify the peak corresponding to the indole NH of W14 in ATI1-N and ATI2-N (Figure 6C). We then recorded spectra in the presence and absence of purified ATG8f expressed in E. coli (Figure 6D,E), and examined the regions of the spectra with chemical shifts from the indole NH protons. This analysis showed clear line broadening of the W14 indole NH signal in the presence of ATG8f (Figure 6E), indicating that binding involves the N-terminal AIM of both ATI1-N and ATI2-N. We conclude that the N-terminal AIMs of ATI1 and ATI2 are necessary and sufficient for binding to ATG8f. We also conclude from the sharp peaks of the indole NH protons in the unbound state that the functional AIMs in both ATI1 and ATI2 reside in regions of high flexibility.
Region including the N-terminal AIM motif (WEVV) at position 14-17.
Protein: DP02549 (UniProt O82775)
Region: residues 14	to 17
Annotation: selective autophagy (Biological process)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
To test directly whether the N-terminal or C-terminal AIMs are functional, we first constructed point mutants in both putative AIMs in ATI2 and performed directed yeast two-hybrid assays with ATG8f. These experiments showed that ATI2 interaction with ATG8f was abolished upon mutation of the putative AIM in the N-terminal IDR, but not upon mutation of the putative AIM in the C-terminal part (Figure 6A). Thus, the N-terminal, but not the C-terminal AIM of ATI2 is necessary for ATG8 interaction in yeast.
Region including the N-terminal AIM motif (WEVV) at position 14-17.
Protein: DP02549 (UniProt O82775)
Region: residues 14	to 17
Annotation: protein binding (Molecular function)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
To test directly whether the N-terminal or C-terminal AIMs are functional, we first constructed point mutants in both putative AIMs in ATI2 and performed directed yeast two-hybrid assays with ATG8f. These experiments showed that ATI2 interaction with ATG8f was abolished upon mutation of the putative AIM in the N-terminal IDR, but not upon mutation of the putative AIM in the C-terminal part (Figure 6A). Thus, the N-terminal, but not the C-terminal AIM of ATI2 is necessary for ATG8 interaction in yeast.
Region including the N-terminal AIM motif (WEVV) at position 14-17.
Protein: DP02550 (UniProt Q8VY98)
Region: residues 1	to 193
Annotation: disorder (Structural state)
Evidence: sodium dodecyl sulfate polyacrylamide gel electrophoresis evidence used in manual assertion
Supporting statements:
We noticed that the migration rates of ATI1-N and ATI2-N in SDS-PAGE were slower than
expected by their molecular weights (MwATI1 -N = 20.46 kDa; MwATI2-N = 21.02 kDa) (Fig. 3A). This simple observation represents a first indication that ATI1 -N and ATI2-N may be intrinsically disordered, because IDRs are typically depleted in hydrophobic residues, and, consequently, tend to bind less SDS, explaining their abnormally slow mobility in SDS-PAGE [37].
Protein: DP02550 (UniProt Q8VY98)
Region: residues 1	to 193
Annotation: disorder (Structural state)
Evidence: temperature-induced protein unfolding evidence used in manual assertion
Supporting statements:
Since we cannot exclude that ATI1-N and ATI2-N form folded dimers, we subjected ATI1-N, ATI2-N and the globular GST to heat treatments for 10 min at 75°C or 5 min at 95°C, and separated aggregated from soluble protein by centrifugation (Figure 3C–D). These aggregation tests showed that, in contrast with the GST control, ATI1-N and ATI2-N remained soluble upon subjection to elevated temperatures that unfold most globular proteins from non-thermophilic organisms.
Protein: DP02550 (UniProt Q8VY98)
Region: residues 1	to 193
Annotation: disorder (Structural state)
Evidence: chromatography evidence used in manual assertion
Supporting statements:
We next subjected protein samples of ATI1-N, ATI2-N and the similarly sized, globular prolactin (PRL, 23 kDa) to size-exclusion chromatography in which the elution volume decreases with the Stokes radius of the protein, such that folded proteins elute at larger volumes than IDPs of similar molecular mass. ATI2-N eluted at the smallest volume (67 ml), followed by ATI1-N (75 ml), and finally the globular PRL (85 ml) (Figure 3B), suggesting that the Stokes radii (r) increase in the order r(PRL) < r(ATI1-N) < r(ATI2-N).
globular PRL (85 ml) (Fig. 3B), suggesting that the Stokes radii (r) increase in the order r(PRL) < r(ATI1-N) < r(ATI2-N).
Protein: DP02550 (UniProt Q8VY98)
Region: residues 1	to 193
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The CD spectra of both ATI1-N and ATI2-N showed characteristic negative minima ∼200 nm, indicating a low content of secondary structure (Figure 4A). In addition, the broad minima at ∼220 nm may indicate some low (<10%) α-helical content [42], in agreement with the secondary structure prediction obtained utilizing the Psipred tool (Figure 4B) [43,44].
Protein: DP02550 (UniProt Q8VY98)
Region: residues 1	to 193
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
ATI2-N shows spectral properties of an IDP in all three regions: methyl protons show a narrow dispersion of chemical shifts above 0.8 ppm, amide protons resonate at chemical shifts within the interval 8.0 < δ < 8.7 ppm, and the three tryptophan NHs in ATI2-N give rise to sharp peaks reflecting high conformational flexibility (Fig. 5). Thus, together with the size exclusion chromatography and CD results, we conclude that ATI2-N is a highly disordered IDP with little or no long-range contacts.
Protein: DP02550 (UniProt Q8VY98)
Region: residues 13	to 18
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
We next used the well-resolved indole NH peaks in the 1D 1H NMR spectra to query whether the ATI1-N and ATI2-N were sufficient for ATG8 interaction. First, we expressed wild type and W14A/V17A mutants of ATI1-N and ATI2-N (Figure 6B), and recorded 1D 1H NMR spectra to identify the peak corresponding to the indole NH of W14 in ATI1-N and ATI2-N (Figure 6C). We then recorded spectra in the presence and absence of purified ATG8f expressed in E. coli (Figure 6D,E), and examined the regions of the spectra with chemical shifts from the indole NH protons. This analysis showed clear line broadening of the W14 indole NH signal in the presence of ATG8f (Figure 6E), indicating that binding involves the N-terminal AIM of both ATI1-N and ATI2-N. We conclude that the N-terminal AIMs of ATI1 and ATI2 are necessary and sufficient for binding to ATG8f. We also conclude from the sharp peaks of the indole NH protons in the unbound state that the functional AIMs in both ATI1 and ATI2 reside in regions of high flexibility.
Region including the N-terminal AIM motif (WEVV) at position 14-17.
Protein: DP02550 (UniProt Q8VY98)
Region: residues 13	to 18
Annotation: selective autophagy (Biological process)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
We next used the well-resolved indole NH peaks in the 1D 1H NMR spectra to query whether the ATI1-N and ATI2-N were sufficient for ATG8 interaction. First, we expressed wild type and W14A/V17A mutants of ATI1-N and ATI2-N (Figure 6B), and recorded 1D 1H NMR spectra to identify the peak corresponding to the indole NH of W14 in ATI1-N and ATI2-N (Figure 6C). We then recorded spectra in the presence and absence of purified ATG8f expressed in E. coli (Figure 6D,E), and examined the regions of the spectra with chemical shifts from the indole NH protons. This analysis showed clear line broadening of the W14 indole NH signal in the presence of ATG8f (Figure 6E), indicating that binding involves the N-terminal AIM of both ATI1-N and ATI2-N. We conclude that the N-terminal AIMs of ATI1 and ATI2 are necessary and sufficient for binding to ATG8f. We also conclude from the sharp peaks of the indole NH protons in the unbound state that the functional AIMs in both ATI1 and ATI2 reside in regions of high flexibility.
Region including the N-terminal AIM motif (WEVV) at position 14-17.
Protein: DP02550 (UniProt Q8VY98)
Region: residues 13	to 18
Annotation: selective autophagy (Biological process)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
To test directly whether the N-terminal or C-terminal AIMs are functional, we first constructed point mutants in both putative AIMs in ATI2 and performed directed yeast two-hybrid assays with ATG8f. These experiments showed that ATI2 interaction with ATG8f was abolished upon mutation of the putative AIM in the N-terminal IDR, but not upon mutation of the putative AIM in the C-terminal part (Figure 6A). Thus, the N-terminal, but not the C-terminal AIM of ATI2 is necessary for ATG8 interaction in yeast.
Region including the N-terminal AIM motif (WEVV) at position 14-17.
Protein: DP02550 (UniProt Q8VY98)
Region: residues 13	to 18
Annotation: protein binding (Molecular function)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
To test directly whether the N-terminal or C-terminal AIMs are functional, we first constructed point mutants in both putative AIMs in ATI2 and performed directed yeast two-hybrid assays with ATG8f. These experiments showed that ATI2 interaction with ATG8f was abolished upon mutation of the putative AIM in the N-terminal IDR, but not upon mutation of the putative AIM in the C-terminal part (Figure 6A). Thus, the N-terminal, but not the C-terminal AIM of ATI2 is necessary for ATG8 interaction in yeast.
Region including the N-terminal AIM motif (WEVV) at position 14-17.
Protein: DP02551 (UniProt Q8NBP7)
Region: residues 31	to 60
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
There are 16 PCSK9 crystal structures deposited in the Protein Data Bank (www.rcsb.org; as of July 2018), which used recombinant PCSK9 with an intact prodomain for crystallization (PDB accession codes 2P4E, 3SQO, 4NE9, 5VL7, 5VLA, 5VLH, 5VLL, 5VLP, 2PMW, 3H42, 3P5B, 3P5C, 4NMX, 5VLK, 2QTW, 4K8R). They comprise apo-structures of full length and of C-terminally truncated PCSK9 and of complexes of PCSK9 with antibody Fabs and with peptidic inhibitors. Despite the different PCSK9 forms used and the different space groups, all structures share the common feature in that there is no recognizable electron density for the N-terminal region of the PCSK9 prodomain encompassing residues Q31–T60. This strongly suggests that this segment is in a disordered state.
Protein: DP02551 (UniProt Q8NBP7)
Region: residues 45	to 49
Annotation: phosphorylation display site (Disorder function)
Evidence: combinatorial experimental and author inference evidence contained in single publication used in manual assertion
Supporting statements:
The second PTM site is S47, which undergoes phosphorylation [30], [31] mediated by the Golgi casein kinase FAM20C [31], and the S47-phosphorylated PCSK9 was detected in human plasma [30]. The S47 phosphorylation appears to protect the acidic stretch from proteolysis [30].