LiteFold/DisProt huggingface dataset:
Protein: DP01189 (UniProt P53874)
Region: residues 1	to 359
Annotation: disorder (Structural state)
Evidence: combinatorial experimental and curator inference evidence used in manual assertion
Supporting statements:
Purified Ubp10's spectrum (Fig. 3C, black line) contains minima at 204 and 218 nm, indicative of the known α-helical structure in its DUB catalytic domain (26) (Fig. 1A). However, when compared with the highly structured α-helical protein BSA (Fig. 3C, gray line), the shift of Ubp10's spectrum downwards and toward 200 nm is characteristic of a protein with a high degree of random coil (31).
Purified Ubp10 or BSA was exposed to trypsin over a time course of 45 min. Ubp10 was rapidly degraded with almost no visible full-length protein after 2 min of exposure to trypsin (Fig. 3D).
The CD and limited proteolysis results in combination with different disorder predictors used in the publication allow us to define this region as disordered.
Protein: DP01189 (UniProt P53874)
Region: residues 730	to 792
Annotation: disorder (Structural state)
Evidence: combinatorial experimental and curator inference evidence used in manual assertion
Supporting statements:
Purified Ubp10's spectrum (Fig. 3C, black line) contains minima at 204 and 218 nm, indicative of the known α-helical structure in its DUB catalytic domain (26) (Fig. 1A). However, when compared with the highly structured α-helical protein BSA (Fig. 3C, gray line), the shift of Ubp10's spectrum downwards and toward 200 nm is characteristic of a protein with a high degree of random coil (31).
Purified Ubp10 or BSA was exposed to trypsin over a time course of 45 min. Ubp10 was rapidly degraded with almost no visible full-length protein after 2 min of exposure to trypsin (Fig. 3D).
The CD and limited proteolysis results in combination with different disorder predictors used in the publication allow us to define this region as disordered.
Protein: DP01189 (UniProt P53874)
Region: residues 2	to 27
Annotation: protein binding (Molecular function)
Evidence: yeast 2-hybrid evidence used in manual assertion
Supporting statements:
We found that regions of Ubp10 required to interact with each protein were also sufficient for that interaction (Fig. 4C). Residues 2–27 of Ubp10 solely interacted with Dhr2. Residues 109–145 of Ubp10 solely interacted with Sir4. Residues 167–208 of Ubp10 solely interacted with Utp22.
Protein: DP01189 (UniProt P53874)
Region: residues 109	to 145
Annotation: protein binding (Molecular function)
Evidence: yeast 2-hybrid evidence used in manual assertion
Supporting statements:
We found that regions of Ubp10 required to interact with each protein were also sufficient for that interaction (Fig. 4C). Residues 2–27 of Ubp10 solely interacted with Dhr2. Residues 109–145 of Ubp10 solely interacted with Sir4. Residues 167–208 of Ubp10 solely interacted with Utp22.
Protein: DP01189 (UniProt P53874)
Region: residues 167	to 208
Annotation: protein binding (Molecular function)
Evidence: yeast 2-hybrid evidence used in manual assertion
Supporting statements:
We found that regions of Ubp10 required to interact with each protein were also sufficient for that interaction (Fig. 4C). Residues 2–27 of Ubp10 solely interacted with Dhr2. Residues 109–145 of Ubp10 solely interacted with Sir4. Residues 167–208 of Ubp10 solely interacted with Utp22.
Protein: DP01189 (UniProt P53874)
Region: residues 171	to 208
Annotation: protein binding (Molecular function)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
Residues 2–27 in Ubp10 were required to interact with Dhr2, and loss of these residues did not affect the interaction with Sir4 or Utp22. Likewise, Ubp10 required residues 109–122 and 123–141 to interact with Sir4, and loss of these residues had no effect on interaction with Dhr2 and Utp22. Finally, Ubp10 required residues 171–208 to interact with Utp22, and loss of these residues had no effect on interaction with Dhr2 and Sir4. Thus, each of the three representative Ubp10 interactions is defined by its own unique binding module within Ubp10's N-terminal IDR.
Protein: DP01189 (UniProt P53874)
Region: residues 109	to 141
Annotation: protein binding (Molecular function)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
Residues 2–27 in Ubp10 were required to interact with Dhr2, and loss of these residues did not affect the interaction with Sir4 or Utp22. Likewise, Ubp10 required residues 109–122 and 123–141 to interact with Sir4, and loss of these residues had no effect on interaction with Dhr2 and Utp22. Finally, Ubp10 required residues 171–208 to interact with Utp22, and loss of these residues had no effect on interaction with Dhr2 and Sir4. Thus, each of the three representative Ubp10 interactions is defined by its own unique binding module within Ubp10's N-terminal IDR.
Protein: DP01189 (UniProt P53874)
Region: residues 2	to 27
Annotation: protein binding (Molecular function)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
Residues 2–27 in Ubp10 were required to interact with Dhr2, and loss of these residues did not affect the interaction with Sir4 or Utp22. Likewise, Ubp10 required residues 109–122 and 123–141 to interact with Sir4, and loss of these residues had no effect on interaction with Dhr2 and Utp22. Finally, Ubp10 required residues 171–208 to interact with Utp22, and loss of these residues had no effect on interaction with Dhr2 and Sir4. Thus, each of the three representative Ubp10 interactions is defined by its own unique binding module within Ubp10's N-terminal IDR.
Protein: DP01189 (UniProt P53874)
Region: residues 109	to 141
Annotation: subtelomeric heterochromatin assembly (Biological process)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
Only the Ubp10 mutant lacking the residues required for Sir4 interaction resulted in loss of telomere chromatin silencing equivalent to the loss of silencing resulting from deletion of UBP10 (ubp10Δ) (Fig. 4D). Thus, the Ubp10-binding module for Sir4 is not only required to coordinate the Ubp10-Sir4 interaction, but is also required to direct Ubp10's functional role in telomere chromatin silencing.
Protein: DP01191 (UniProt O14140)
Region: residues 1	to 54
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Figure 2D. Calpha secondary chemical shifts of Dss1 confirm the predominantly disordered structure.
Protein: DP01191 (UniProt O14140)
Region: residues 1	to 54
Annotation: disorder to order (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Dss1 is intrinsically disordered with a single, transiently populated alpha helix from F55 through K66.
Protein: DP01191 (UniProt O14140)
Region: residues 1	to 54
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Titration analyses with increasing amounts of ubiquitin disclosed the strongest binding to ubiquitin by binding site I (UBS-I), which is located at D38-D49 and disclosed the second and weakest site, UBS-II, located at D16-N25.
Protein: DP01191 (UniProt O14140)
Region: residues 1	to 54
Annotation: pre-molten globule (Structural state)
Evidence: heteronuclear single quantum coherence spectroscopy evidence used in manual assertion
Supporting statements:
Comparison of the NMR peak intensities of 15N-Dss1-N71C with and without MTSL indicated transient long-range effects from the C-terminal spin label (N71C) to regions >30 residues away. Thus, the C terminus was observed to be close to binding site I (BS-I) as well as to the linker between BS-I and BS-II (Figures 1C and S1C).
Because the PREs, the chemical shifts effects, and the population of the helix are all less than maximal, the helix of Dss1 is not tightly anchored but rather forms a “cloud” that dynamically shields access to the helix itself and to BS-I (Figure 1E). Such equilibrium between open and closed conformations may have consequences for how, and to which extent, the binding sites in Dss1 are available.
Protein: DP01191 (UniProt O14140)
Region: residues 1	to 54
Annotation: molecular adaptor activity (Molecular function)
Evidence: liquid chromatography coupled with tandem mass spectrometry evidence used in manual assertion
Supporting statements:
For wild-type Dss1, GFP-Dss1 preparations were enriched in 263 proteins (Figure S3B).
Comparison of the GFP-Dss1 and GFP-Dss1Δhelix interactomes revealed that most interactions (Figures S3D–S3F) occurred independently of the helix (Figure 2D, green points). This agrees with previous reports that most binding to Dss1 is confined to the disordered region (Kragelund et al., 2016).
Protein: DP01191 (UniProt O14140)
Region: residues 1	to 54
Annotation: calcium ion binding (Molecular function)
Evidence: heteronuclear single quantum coherence spectroscopy evidence used in manual assertion
Supporting statements:
We included the highly multispecific yeast protein DSS1wt, which has a high κ-value, indicating regions of repetitive charged residues of the same charge. In this case, we found two regions of higher CSPs, one between residues S8 to D14, and one between residues T39 to N43 (Figure 1D). Although these regions had the highest CSP, we observed generally high CSPs throughout the protein, i.e., S8–D14, D18–E32, and T39–N43.
Protein: DP01191 (UniProt O14140)
Region: residues 1	to 54
Annotation: calcium ion binding (Molecular function)
Evidence: small molecule detection assay evidence used in manual assertion
Supporting statements:
DSS1wt binds calcium at a higher ratio of 1:4 (Figure 1G,I), which may cause overlap within the regions with high CSPs. The overall affinity of DSS1wt for calcium was measured to be ~70 μM (Figure 1H).
Protein: DP01191 (UniProt O14140)
Region: residues 1	to 54
Annotation: disorder to pre-molten globule (Structural transition)
Evidence: nuclear magnetic resonance evidence used in manual assertion
Supporting statements:
We next acquired DOSY spectra for DSS1wt and its mutants, finding that both DSS1wt and DSS1D became more compact upon calcium binding (25.7% and 17.6%, respectively).
Protein: DP01192 (UniProt P55036)
Region: residues 246	to 256
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
S5a (196-306) contains three a-helices that are connected by flexible regions.
S5a (196-306) contains three well-structured regions spanning residues P214-E245, D257-E269, and L278-Q296 (Figure 1(b)–(e)). Flexible, randomly coiled linker regions connect the structured elements and prevent them from being defined relative to each other. This lack of higher-order structure within these regions is supported by their chemical-shift assignments,26 lack of long-range nuclear Overhauser effect (NOE) interactions, and NMR relaxation data (data not shown).
Between the UIMs of S5a is a second α-helix with flexible linker regions at either end (Figure 1).
Cross-references: PDB:1YX4
Protein: DP01192 (UniProt P55036)
Region: residues 246	to 256
Annotation: flexible linker (Disorder function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
S5a (196-306) contains three well-structured regions spanning residues P214-E245, D257-E269, and L278-Q296 (Figure 1(b)–(e)). Flexible, randomly coiled linker regions connect the structured elements and prevent them from being defined relative to each other. This lack of higher-order structure within these regions is supported by their chemical-shift assignments,26 lack of long-range nuclear Overhauser effect (NOE) interactions, and NMR relaxation data (data not shown).
Between the UIMs of S5a is a second α-helix with flexible linker regions at either end (Figure 1).
Protein: DP01192 (UniProt P55036)
Region: residues 305	to 377
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Here, we identify a region at the C-terminal end of hRpn10 that forms a binding site in the proteasome for E6AP. By using biophysical techniques including NMR spectroscopy, we find this region to be disordered when unbound
Cross-references: PDB:6U19
Protein: DP01192 (UniProt P55036)
Region: residues 332	to 341
Annotation: disorder to order (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Comparisons of our NMR data acquired on free and AZUL-bound RAZUL indicate that RAZUL acquires helicity upon binding to AZUL. Carbonyl and Cα values when compared with random coil taking into account amino acid type yields a chemical shift index (CSI) that informs on secondary structure; these values shift to reflect greater helicity for RAZUL when bound to AZUL (Fig. 5c and Supplementary Fig. 6b). Moreover, intramolecular interactions characteristic of helicity were observed following AZUL addition, but not for free RAZUL (Supplementary Fig. 6c, d). Overall, our NMR data indicate that RAZUL switches from a poorly ordered state to a well-defined helical state following AZUL binding.
Cross-references: PDB:6U19
Protein: DP01192 (UniProt P55036)
Region: residues 328	to 361
Annotation: molecular adaptor activity (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
RAZUL binds the AZUL helices from the opposite direction compared with the Zn finger. In this complex, two α-helices are formed in RAZUL that span P332–N341 (α1) and E350–S361 (α2). Directly N-terminal to RAZUL α1 is a single turn of a 3 10 -helix that spans V328-Q330.
RAZUL α1 is centered between the two AZUL helices by hydrophobic interactions involving F334, L335, V338, and L339 as well as L342 and V345 from the RAZUL α1/ α2 loop. From the 3 10 -helix, V328 and M329 form hydrophobic interactions. RAZUL α2 is more peripheral compared with α1, with A351, I352, A355, M356, and L359.
Cross-references: PDB:6U19
Protein: DP01192 (UniProt P55036)
Region: residues 328	to 361
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
E6AP AZUL binds to hRpn10 RAZUL at the proteasome.
Cross-references: PDB:6U19
Protein: DP01192 (UniProt P55036)
Region: residues 350	to 361
Annotation: disorder to order (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Comparisons of our NMR data acquired on free and AZUL-bound RAZUL indicate that RAZUL acquires helicity upon binding to AZUL. Carbonyl and Cα values when compared with random coil taking into account amino acid type yields a chemical shift index (CSI) that informs on secondary structure; these values shift to reflect greater helicity for RAZUL when bound to AZUL (Fig. 5c and Supplementary Fig. 6b). Moreover, intramolecular interactions characteristic of helicity were observed following AZUL addition, but not for free RAZUL (Supplementary Fig. 6c, d). Overall, our NMR data indicate that RAZUL switches from a poorly ordered state to a well-defined helical state following AZUL binding.
Cross-references: PDB:6U19
Protein: DP01192 (UniProt P55036)
Region: residues 305	to 377
Annotation: disorder to order (Structural transition)
Evidence: circular dichroism evidence used in manual assertion
Supporting statements:
CD measurements indicated 7% and 46% helicity, respectively, for unbound RAZUL (blue) and AZUL (green), and a theoretical spectrum (gray dashed line) for the mixture with markedly less spectral features of helicity compared with the recorded experimental spectrum (orange, Fig. 5d). Overall, 35% helicity is indicated from the experimental CD data recorded on the complex, consistent with the 36% helicity determined by NMR (Fig. 5b). The AZUL secondary structure is unaltered by binding to RAZUL (Supplementary Fig. 6a), leading us to conclude that the observed difference between the theoretical and experimental CD spectra reflects increased helicity for RAZUL, consistent with the NMR data (for example, Fig. 5c).
Protein: DP01192 (UniProt P55036)
Region: residues 305	to 377
Annotation: disorder (Structural state)
Evidence: circular dichroism evidence used in manual assertion
Supporting statements:
CD measurements indicated 7% and 46% helicity, respectively, for unbound RAZUL (blue) and AZUL (green), and a theoretical spectrum (gray dashed line) for the mixture with markedly less spectral features of helicity compared with the recorded experimental spectrum (orange, Fig. 5d). Overall, 35% helicity is indicated from the experimental CD data recorded on the complex, consistent with the 36% helicity determined by NMR (Fig. 5b). The AZUL secondary structure is unaltered by binding to RAZUL (Supplementary Fig. 6a), leading us to conclude that the observed difference between the theoretical and experimental CD spectra reflects increased helicity for RAZUL, consistent with the NMR data (for example, Fig. 5c).
Protein: DP01192 (UniProt P55036)
Region: residues 305	to 377
Annotation: molecular adaptor activity (Molecular function)
Evidence: circular dichroism evidence used in manual assertion
Supporting statements:
CD measurements indicated 7% and 46% helicity, respectively, for unbound RAZUL (blue) and AZUL (green), and a theoretical spectrum (gray dashed line) for the mixture with markedly less spectral features of helicity compared with the recorded experimental spectrum (orange, Fig. 5d). Overall, 35% helicity is indicated from the experimental CD data recorded on the complex, consistent with the 36% helicity determined by NMR (Fig. 5b). The AZUL secondary structure is unaltered by binding to RAZUL (Supplementary Fig. 6a), leading us to conclude that the observed difference between the theoretical and experimental CD spectra reflects increased helicity for RAZUL, consistent with the NMR data (for example, Fig. 5c).
Protein: DP01192 (UniProt P55036)
Region: residues 305	to 377
Annotation: protein binding (Molecular function)
Evidence: co-immunoprecipitation evidence used in manual assertion
Supporting statements:
Expression of full-length (lane 7) but not RAZUL-truncated hRpn10 (lane 6) resulted in observable E6AP co-immunoprecipitation with proteasomes isolated from ΔRAZUL cells (Fig. 4a); we attribute the lower amounts of E6AP co-immunoprecipitated with proteasomes of hRpn10full-length-expressing ΔRAZUL cells to the reduced abundance of endogenous E6AP in this cell line, as described above (Fig. 2b). This experiment indicates that the hRpn10 RAZUL recruits E6AP to the proteasome.
Protein: DP01192 (UniProt P55036)
Region: residues 305	to 377
Annotation: protein binding (Molecular function)
Evidence: isothermal titration calorimetry evidence used in manual assertion
Supporting statements:
To assess the strength of hRpn10(305–377):AZUL interaction, we used isothermal titration calorimetry (ITC) with the AZUL added incrementally to hRpn10(305–377); a Kd value of 11.6 + 3.3 nM was measured (Fig. 1d and Supplementary Fig. 2f), indicating similar strength to hRpn13 interaction with the proteasome.
Protein: DP01192 (UniProt P55036)
Region: residues 305	to 377
Annotation: protein binding (Molecular function)
Evidence: surface plasmon resonance evidence used in manual assertion
Supporting statements:
Surface plasmon resonance (SPR) similarly revealed a Kd value of 8.1 + 1.4 nM for GST-hRpn10(305–377) binding to AZUL (Fig. 1d and Supplementary Fig. 2g).
Protein: DP01192 (UniProt P55036)
Region: residues 305	to 377
Annotation: protein binding (Molecular function)
Evidence: circular dichroism evidence used in manual assertion
Supporting statements:
The AZUL secondary structure is unaltered by binding to RAZUL (Supplementary Fig. 6a), leading us to conclude that the observed difference between the theoretical and experimental CD spectra reflects increased helicity for RAZUL, consistent with the NMR data (for example, Fig. 5c).
Protein: DP01193 (UniProt Q9H211)
Region: residues 92	to 159
Annotation: disorder (Structural state)
Evidence: combinatorial experimental and author inference evidence contained in single publication used in manual assertion
Supporting statements:
We used Dynamic Light Scattering (DLS) to estimate the radius of hydration (R(h)) as well as polydispersity of Cdt1 in solution. Ovalbumin showed similar R(h) values in our system as with published results (60). For Cdt1, DLS demonstrated the protein was 99% monomeric, had a calculated R(h) of 3.7 nm, and was 40% polydisperse (Figure 3A). This relatively high level of polydispersity is consistent with a mixed-folded protein with many different conformations and also is consistent with thermally instability at relatively low temperatures (61).
We used both circular dichroism (CD) and intrinsic fluorescence (Ex280, Em330/350) to measure the changes in secondary and tertiary elements, respectively. Our thermal stability data for ovalbumin (Figure 2B & 2C) was similar to previously published data (48) given the difference in buffers (Table 1 & 2). For both DSF and DSCD, Cdt1 followed a simple two-state unfolding process. In fluorescence detection, we observed a melting temperature (Tm) of 43 °C (Figure 2B). In CD, we calculated a Tm of 42 °C (Figure 2C).
We also performed 1H/15N HSQC NMR to validate our CD results. The number of peaks, their broadness and their location similarly demonstrate a high disorder content (56). This data confirms all but the WH domains are disordered in human Cdt1.
Using our experimental secondary structure content analysis, we defined the N-terminal and linker regions as entirely flexible.
The authors define the WH domains as those spanning the residues 159-356 and 410-546.
Protein: DP01193 (UniProt Q9H211)
Region: residues 356	to 410
Annotation: disorder (Structural state)
Evidence: combinatorial experimental and author inference evidence contained in single publication used in manual assertion
Supporting statements:
We used Dynamic Light Scattering (DLS) to estimate the radius of hydration (R(h)) as well as polydispersity of Cdt1 in solution. Ovalbumin showed similar R(h) values in our system as with published results (60). For Cdt1, DLS demonstrated the protein was 99% monomeric, had a calculated R(h) of 3.7 nm, and was 40% polydisperse (Figure 3A). This relatively high level of polydispersity is consistent with a mixed-folded protein with many different conformations and also is consistent with thermally instability at relatively low temperatures (61).
We used both circular dichroism (CD) and intrinsic fluorescence (Ex280, Em330/350) to measure the changes in secondary and tertiary elements, respectively. Our thermal stability data for ovalbumin (Figure 2B & 2C) was similar to previously published data (48) given the difference in buffers (Table 1 & 2). For both DSF and DSCD, Cdt1 followed a simple two-state unfolding process. In fluorescence detection, we observed a melting temperature (Tm) of 43 °C (Figure 2B). In CD, we calculated a Tm of 42 °C (Figure 2C).
We also performed 1H/15N HSQC NMR to validate our CD results. The number of peaks, their broadness and their location similarly demonstrate a high disorder content (56). This data confirms all but the WH domains are disordered in human Cdt1.
Using our experimental secondary structure content analysis, we defined the N-terminal and linker regions as entirely flexible.
The authors define the WH domains as those spanning the residues 159-356 and 410-546.