LiteFold/DisProt huggingface dataset:
Protein: DP04149 (UniProt P51788)
Region: residues 14	to 28
Annotation: self-assembly (Disorder function)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The lack of sufficient density for modeling residues 1–87 suggests that the N terminal cytoplasmic region of CLC-2 is intrinsically disordered. However, our observation of a resolvable hairpin density at the intracellular pore, together with the previous hypothesis of N-terminal ball-and-chain gating for CLC-2, prompted us to consider whether the hairpin density corresponds to N-terminal residues. We tested several candidate fragments from the CLC-2 N-terminal region and found that the hairpin density fits perfectly with residues corresponding to 14–28 (Figure 5A). Inspection of the Q-score validation plot for these residues shows they have higher values than the Q-score expected for the corresponding resolution (Figure 5B), which indicates reliable model building at this resolution (Pintilie et al., 2020).
Along with the absence of hairpin density, the subunit with the rotated CTD exhibits a difference in the linker connecting TM helices J and K, which is poorly resolved and insufficient for model building. In contrast, the subunits with the hairpin density contain reliable density in the JK-linker region, allowing confident modeling of an alpha helix (‘JK-helix’, Figure 5A). This observation suggests that the JK-helix stabilizes the N-terminal hairpin peptide. Indeed, residues on the JK-helix interact directly with residues at the peptide’s hairpin turn (Figure 5C). Interactions with other regions in the TM domain (near helices C, D, J, and R) appear to further stabilize the hairpin in its binding site. In addition, the negative surface potential of the hairpin likely contributes to its interaction with the positive TM domain (Figure 5—figure supplement 3; Video 5).
Cross-references: PDB:8TA4
PDB:8TA5
EMDB:EMD-41129
EMDB:EMD-41128
Protein: DP04149 (UniProt P51788)
Region: residues 14	to 27
Annotation: self-inhibition (Disorder function)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
The hairpin structure appears to directly block the pore (Figure 5—figure supplement 5). To evaluate the potential functional effect of pore block by the N-terminal hairpin, we performed whole-cell patch-clamp recording on CLC-2, both WT and a deletion mutant lacking the hairpin residues (Delta-N), expressed in CHO cells. Delta-N CLC-2 exhibits currents that display less rectification and faster activation kinetics than WT CLC-2 (Figure 6A, B, Figure 6—figure supplement 1).
First, we demonstrate that the increase in current in hairpin-deleted channels reflects an increase in channel activity rather than an increase in surface expression (Figure 6D, E), consistent with loss of a channel-closing mechanism.
Protein: DP04149 (UniProt P51788)
Region: residues 14	to 27
Annotation: chloride channel regulator activity (Molecular function)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
The hairpin structure appears to directly block the pore (Figure 5—figure supplement 5). To evaluate the potential functional effect of pore block by the N-terminal hairpin, we performed whole-cell patch-clamp recording on CLC-2, both WT and a deletion mutant lacking the hairpin residues (Delta-N), expressed in CHO cells. Delta-N CLC-2 exhibits currents that display less rectification and faster activation kinetics than WT CLC-2 (Figure 6A, B, Figure 6—figure supplement 1).
First, we demonstrate that the increase in current in hairpin-deleted channels reflects an increase in channel activity rather than an increase in surface expression (Figure 6D, E), consistent with loss of a channel-closing mechanism.
Protein: DP04149 (UniProt P51788)
Region: residues 14	to 28
Annotation: order (Structural state)
Evidence: cryogenic electron microscopy evidence used in manual assertion
Supporting statements:
The lack of sufficient density for modeling residues 1–87 suggests that the N terminal cytoplasmic region of CLC-2 is intrinsically disordered. However, our observation of a resolvable hairpin density at the intracellular pore, together with the previous hypothesis of N-terminal ball-and-chain gating for CLC-2, prompted us to consider whether the hairpin density corresponds to N-terminal residues. We tested several candidate fragments from the CLC-2 N-terminal region and found that the hairpin density fits perfectly with residues corresponding to 14–28 (Figure 5A). Inspection of the Q-score validation plot for these residues shows they have higher values than the Q-score expected for the corresponding resolution (Figure 5B), which indicates reliable model building at this resolution (Pintilie et al., 2020).
Cross-references: PDB:8TA5
PDB:8TA4
EMDB:EMD-41128
EMDB:EMD-41129
Protein: DP04149 (UniProt P51788)
Region: residues 409	to 418
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy evidence used in manual assertion
Supporting statements:
The cryo-EM structures of CLC-2 chloride channel transmembrane domain with octh symmetric and asymmetric C-terminal regions show this region is disordered.
Cross-references: PDB:8TA5
PDB:8TA4
EMDB:EMD-41128
EMDB:EMD-41129
Protein: DP04149 (UniProt P51788)
Region: residues 14	to 28
Annotation: disorder to order (Structural transition)
Evidence: curator inference from database used in manual assertion
Supporting statements:
The lack of sufficient density for modeling residues 1–87 suggests that the N terminal cytoplasmic region of CLC-2 is intrinsically disordered. However, our observation of a resolvable hairpin density at the intracellular pore, together with the previous hypothesis of N-terminal ball-and-chain gating for CLC-2, prompted us to consider whether the hairpin density corresponds to N-terminal residues. We tested several candidate fragments from the CLC-2 N-terminal region and found that the hairpin density fits perfectly with residues corresponding to 14–28 (Figure 5A). Inspection of the Q-score validation plot for these residues shows they have higher values than the Q-score expected for the corresponding resolution (Figure 5B), which indicates reliable model building at this resolution (Pintilie et al., 2020).
The cryo-EM structure of the Full length human CLC-2 channel in apo state (PDB 7XF5, pmid:37296152) shows the 1-88 region is fully disordered when there are no Chloride ion present in the sample. However structures obtained with Chloride show this region gains electron density by self associating in a hairpin and interacting with the TM domain of the protein.
Cross-references: PDB:8TA5
PDB:8TA4
EMDB:EMD-41128
EMDB:EMD-41129
PDB:7XF5
EMDB:EMD-33169
Protein: DP04151 (UniProt O49255)
Region: residues 162	to 268
Annotation: disorder (Structural state)
Evidence: sodium dodecyl sulfate polyacrylamide gel electrophoresis evidence used in manual assertion
Supporting statements:
Although the relative migration pattern of the recombinant proteins by SDS/PAGE was in accordance with their relative molecular mass values [NAP (162–268): 15.0 kDa; ANAC019 (163–317): 17.2 kDa; ANAC046 (172–338): 18.2 kDa; histidine–NTL8 (156–335): 20.3 kDa; histidine–SOG (210–449): 29.0 kDa; ANAC013 (161–498): 37.5 kDa], they displayed slow migration during SDS/PAGE compared with standard globular proteins, which is in accordance with their ID nature and previous analysis of two NAC TRDs [39].
Protein: DP04151 (UniProt O49255)
Region: residues 162	to 268
Annotation: pre-molten globule (Structural state)
Evidence: gel-filtration evidence used in manual assertion
Supporting statements:
This aspect was analysed by size-exclusion chromatography for the four untagged NAC TRDs. The hydrodynamic radii (Stokes radii) of ANAC046 (172–338), NAP (162–268) and ANAC019 (163–317) corresponded to pre-molten globule states, whereasANAC013 (161–498) had a more compact structure consistent with its disorder profile (Figures 1d and 3).
Protein: DP04151 (UniProt O49255)
Region: residues 162	to 268
Annotation: pre-molten globule (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Although the spectra of the six recombinant proteins showed different characteristics, they all had a minimum around 200 nm and only slightly negative ellipticities at 222 nm (Figure 4a). Both the [θ]222 and [θ]200 values suggested that histidine–NTL8 (156–335), ANAC013 (161–498), NAP (162–268) and ANAC019 (163–317) are similar, populating pre-molten globule like ensembles, whereas the values for histidine–SOG1 (210–449) and ANAC046 (172–338) suggest more random coil-like ensembles.
Protein: DP04151 (UniProt O49255)
Region: residues 206	to 226
Annotation: transcription regulator activator activity (Molecular function)
Evidence: beta galactosidase functional complementation evidence used in manual assertion
Supporting statements:
Both full-length NAP and its TRD, NAP (163–268), activated
transcription in yeast.
This revealed that the ability to activate transcription was retained until the region with the MEME motif was removed, after which no activity was detected. Similar results were obtained from the same C-terminal truncations of full-length NAP, supporting the importance of the MEME motif region for transcriptional activation activity.
Protein: DP04152 (UniProt Q9C932)
Region: residues 163	to 317
Annotation: disorder (Structural state)
Evidence: sodium dodecyl sulfate polyacrylamide gel electrophoresis evidence used in manual assertion
Supporting statements:
Although the relative migration pattern of the recombinant proteins by SDS/PAGE was in accordance with their relative molecular mass values [NAP (162–268): 15.0 kDa; ANAC019 (163–317): 17.2 kDa; ANAC046 (172–338): 18.2 kDa; histidine–NTL8 (156–335): 20.3 kDa; histidine–SOG (210–449): 29.0 kDa; ANAC013 (161–498): 37.5 kDa], they displayed slow migration during SDS/PAGE compared with standard globular proteins, which is in accordance with their ID nature and previous analysis of two NAC TRDs [39].
Protein: DP04152 (UniProt Q9C932)
Region: residues 163	to 317
Annotation: pre-molten globule (Structural state)
Evidence: gel-filtration evidence used in manual assertion
Supporting statements:
This aspect was analysed by size-exclusion chromatography for the four untagged NAC TRDs. The hydrodynamic radii (Stokes radii) of ANAC046 (172–338), NAP (162–268) and ANAC019 (163–317) corresponded to pre-molten globule states, whereasANAC013 (161–498) had a more compact structure consistent with its disorder profile (Figures 1d and 3).
Protein: DP04152 (UniProt Q9C932)
Region: residues 163	to 317
Annotation: pre-molten globule (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Although the spectra of the six recombinant proteins showed different characteristics, they all had a minimum around 200 nm and only slightly negative ellipticities at 222 nm (Figure 4a). Both the [θ]222 and [θ]200 values suggested that histidine–NTL8 (156–335), ANAC013 (161–498), NAP (162–268) and ANAC019 (163–317) are similar, populating pre-molten globule like ensembles, whereas the values for histidine–SOG1 (210–449) and ANAC046 (172–338) suggest more random coil-like ensembles.
Protein: DP04153 (UniProt Q6NQK2)
Region: residues 210	to 449
Annotation: disorder (Structural state)
Evidence: sodium dodecyl sulfate polyacrylamide gel electrophoresis evidence used in manual assertion
Supporting statements:
Although the relative migration pattern of the recombinant proteins by SDS/PAGE was in accordance with their relative molecular mass values [NAP (162–268): 15.0 kDa; ANAC019 (163–317): 17.2 kDa; ANAC046 (172–338): 18.2 kDa; histidine–NTL8 (156–335): 20.3 kDa; histidine–SOG (210–449): 29.0 kDa; ANAC013 (161–498): 37.5 kDa], they displayed slow migration during SDS/PAGE compared with standard globular proteins, which is in accordance with their ID nature and previous analysis of two NAC TRDs [39].
Protein: DP04153 (UniProt Q6NQK2)
Region: residues 210	to 449
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Although the spectra of the six recombinant proteins showed different characteristics, they all had a minimum around 200 nm and only slightly negative ellipticities at 222 nm (Figure 4a). Both the [θ]222 and [θ]200 values suggested that histidine–NTL8 (156–335), ANAC013 (161–498), NAP (162–268) and ANAC019 (163–317) are similar, populating pre-molten globule like ensembles, whereas the values for histidine–SOG1 (210–449) and ANAC046 (172–338) suggest more random coil-like ensembles.
Protein: DP04154 (UniProt Q9XIN7)
Region: residues 156	to 335
Annotation: disorder (Structural state)
Evidence: sodium dodecyl sulfate polyacrylamide gel electrophoresis evidence used in manual assertion
Supporting statements:
Although the relative migration pattern of the recombinant proteins by SDS/PAGE was in accordance with their relative molecular mass values [NAP (162–268): 15.0 kDa; ANAC019 (163–317): 17.2 kDa; ANAC046 (172–338): 18.2 kDa; histidine–NTL8 (156–335): 20.3 kDa; histidine–SOG (210–449): 29.0 kDa; ANAC013 (161–498): 37.5 kDa], they displayed slow migration during SDS/PAGE compared with standard globular proteins, which is in accordance with their ID nature and previous analysis of two NAC TRDs [39].
Protein: DP04154 (UniProt Q9XIN7)
Region: residues 156	to 335
Annotation: pre-molten globule (Structural state)
Evidence: sodium dodecyl sulfate polyacrylamide gel electrophoresis evidence used in manual assertion
Supporting statements:
Although the spectra of the six recombinant proteins showed different characteristics, they all had a minimum around 200 nm and only slightly negative ellipticities at 222 nm (Figure 4a). Both the [θ]222 and [θ]200 values suggested that histidine–NTL8 (156–335), ANAC013 (161–498), NAP (162–268) and ANAC019 (163–317) are similar, populating pre-molten globule like ensembles, whereas the values for histidine–SOG1 (210–449) and ANAC046 (172–338) suggest more random coil-like ensembles.
Protein: DP04155 (UniProt P28749)
Region: residues 612	to 687
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The 2D [1H,15N] HSQC spectrum of 15N-labeled p107 (residues M612-S687, which include R1, R2, and R3) shows all hallmarks of an intrinsically disordered protein (IDP), with a highly limited proton chemical shift dispersion due to the lack of a hydrogen bond network in secondary structure elements.
Cross-references: BMRB:28091
Protein: DP04155 (UniProt P28749)
Region: residues 614	to 624
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
An overlay of the 2D [1H,15N] HSQC spectrum of 15N-labeled p107M612-S687 in the presence and absence of purified B55α shows that ~15 peaks have reduced intensity, typical for an IDP:protein interaction (Figure 1E and F). Upon completion of the sequence-specific backbone assignment of p107M612-S687, we identified that cross-peaks corresponding to p107 residues M614-E624 were most significantly broadened upon binding to B55α (Figure 1F).
Cross-references: BMRB:28091
Protein: DP04155 (UniProt P28749)
Region: residues 615	to 630
Annotation: protein binding (Molecular function)
Evidence: glutathione S-transferase pull-down assay evidence used in manual assertion
Supporting statements:
To determine the contribution of each conserved region, we generated p107 deletion mutants and performed GST pull-down assays using U-2 OS whole-cell lysates. Figure 1D shows that a mutant lacking residues C-terminal of R2 binds B55α similarly to the full construct, indicating that residues C-terminal to the R2 domain are dispensable for B55α binding. R1 alone can bind B55α, although to a slightly lesser extent than R1/R2 (lane 3), while mutants lacking regions containing R1 do not bind B55α (lanes 6 and 7). Additional experiments highlighted that R1 is the key B55α binding region and that R2 enhances the interaction, but without R1 cannot recruit B55α alone (Figure 1D).
The authors have previously specified on Figure 1 panel C that Region 1 (R1) corresponds to residues 615-630 of p107.
Protein: DP04155 (UniProt P28749)
Region: residues 615	to 630
Annotation: protein binding (Molecular function)
Evidence: point mutation phenotypic evidence used in manual assertion
Supporting statements:
It has been shown that charged:charged interactions are central for B55α:TAU substrate recruitment. To test if charged:charged interactions are also important for p107 recruitment, we generated p107 R621A and K623A variants (in R1), R633A and R34A variants (in s2), and an R647A variant (in R2) using GST-p107-R1R2. As shown in Figure 1D, positive residues in R1 and R2, but not the connecting s2 ‘linker,’ led to a reduction in binding to B55α. Mutation of the cyclin A binding site in R2 (659KRRL-AAAA and 660RRL-AAA mutations to Ala), which also contains positive residues, also reduced binding (data not shown). Thus, positively charged residues participate in binding to B55α.
The authors have previously specified on Figure 1 panel C that Region 1 (R1) corresponds to residues 615-630 of p107.
Protein: DP04155 (UniProt P28749)
Region: residues 647	to 663
Annotation: protein binding (Molecular function)
Evidence: point mutation phenotypic evidence used in manual assertion
Supporting statements:
It has been shown that charged:charged interactions are central for B55α:TAU substrate recruitment. To test if charged:charged interactions are also important for p107 recruitment, we generated p107 R621A and K623A variants (in R1), R633A and R34A variants (in s2), and an R647A variant (in R2) using GST-p107-R1R2. As shown in Figure 1D, positive residues in R1 and R2, but not the connecting s2 ‘linker,’ led to a reduction in binding to B55α. Mutation of the cyclin A binding site in R2 (659KRRL-AAAA and 660RRL-AAA mutations to Ala), which also contains positive residues, also reduced binding (data not shown). Thus, positively charged residues participate in binding to B55α.
The authors have previously specified on Figure 1 panel C that Region 2 (R2) corresponds to residues 647-663 of p107.
Protein: DP04155 (UniProt P28749)
Region: residues 647	to 663
Annotation: protein binding (Molecular function)
Evidence: glutathione S-transferase pull-down assay evidence used in manual assertion
Supporting statements:
To determine the contribution of each conserved region, we generated p107 deletion mutants and performed GST pull-down assays using U-2 OS whole-cell lysates. Figure 1D shows that a mutant lacking residues C-terminal of R2 binds B55α similarly to the full construct, indicating that residues C-terminal to the R2 domain are dispensable for B55α binding. R1 alone can bind B55α, although to a slightly lesser extent than R1/R2 (lane 3), while mutants lacking regions containing R1 do not bind B55α (lanes 6 and 7). Additional experiments highlighted that R1 is the key B55α binding region and that R2 enhances the interaction, but without R1 cannot recruit B55α alone (Figure 1D). Conversely, binding of p107 to CDK2 (or cyclin A, not shown) strictly depends on the presence of R2 (lanes 2, 4, 5, and 6), which includes an RxL motif necessary for cyclin A/CDK2 binding (Adams et al., 1996; Chen et al., 1996).
The authors have previously specified on Figure 1 panel C that Region 2 (R2) corresponds to residues 647-663 of p107.
Protein: DP04155 (UniProt P28749)
Region: residues 647	to 663
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
An overlay of the 2D [1H,15N] HSQC spectrum of 15N-labeled p107M612-S687 in the presence and absence of purified B55α shows that ~15 peaks have reduced intensity, typical for an IDP:protein interaction (Figure 1E and F). Upon completion of the sequence-specific backbone assignment of p107M612-S687, we identified that cross-peaks corresponding to p107 residues M614-E624 were most significantly broadened upon binding to B55α (Figure 1F). Consistent with the mutation data, these residues form the core of R1. Furthermore, residues within the linker region and R2 also showed peak intensity attenuations, consistent with additional weaker interactions in R2 contributing and enhancing the interaction of R1.
The authors have previously specified on Figure 1 panel C that Region 2 (R2) corresponds to residues 647-663 of p107.
Protein: DP04155 (UniProt P28749)
Region: residues 613	to 617
Annotation: phosphorylation display site (Disorder function)
Evidence: protein mass spectrometry evidence used in manual assertion
Supporting statements:
To determine which sites on p107 were dephosphorylated, we generated p107 mutants in which only a single ‘SP’ site can be phosphorylated (i.e., S615A-S640A, S615A-S650A, and S640A-S650A), as well as a triple deletion control (S615A-S640A-S650A) (Figure 3B). To validate phosphorylation/dephosphorylation and determine which sites were targeted by PP2A/B55α, we used mass spectrometry (MS). MS identified pS615 and pS640 in CDK2-phosphorylated p107 and a significant reduction of pS615 and pS640 phosphorylation upon incubation with PP2A/B55α (Figure 3C).
Protein: DP04155 (UniProt P28749)
Region: residues 638	to 642
Annotation: phosphorylation display site (Disorder function)
Evidence: protein mass spectrometry evidence used in manual assertion
Supporting statements:
To determine which sites on p107 were dephosphorylated, we generated p107 mutants in which only a single ‘SP’ site can be phosphorylated (i.e., S615A-S640A, S615A-S650A, and S640A-S650A), as well as a triple deletion control (S615A-S640A-S650A) (Figure 3B). To validate phosphorylation/dephosphorylation and determine which sites were targeted by PP2A/B55α, we used mass spectrometry (MS). MS identified pS615 and pS640 in CDK2-phosphorylated p107 and a significant reduction of pS615 and pS640 phosphorylation upon incubation with PP2A/B55α (Figure 3C).
Protein: DP04155 (UniProt P28749)
Region: residues 615	to 630
Annotation: protein binding (Molecular function)
Evidence: competitive binding evidence used in manual assertion
Supporting statements:
To understand which R1 residues are critical for the p107:B55α interaction, we performed binding competition assays. In this assay, we use increasing concentrations of R1 peptide to compete for B55α binding (Figure 5A). This peptide was able to compete with a p107 R1R2 construct.
The authors have previously specified on Figure 1 panel C that Region 1 (R1) corresponds to residues 615-630 of p107.
Protein: DP04156 (UniProt Q96B97)
Region: residues 163	to 263
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
We accomplished the near-complete assignment of the linker region containing CIN85-PRM1 by acquiring three-dimensional 13C-detected experiments on a shorter construct of the CIN85 protein (CIN85163–333; see Figure S6 for the different protein constructs used in this work and the spectral quality in Figure S7).
Within the disordered linker region (residues 163–263), we were successful in assigning 97% the backbone resonances, including the prolines inside CIN85-PRM1.
Cross-references: BMRB:52081