LiteFold/DisProt huggingface dataset:
Protein: DP01113 (UniProt D0PV95)
Region: residues 1	to 168
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
In contrast, deletion of the RGG-rich N terminus (ΔRGG) results in no observable droplets, even up to concentrations as high as 250 μM, revealing the N-terminal RGG domain as essential for phase separation. Moreover, the isolated N-terminal RGG domain was alone sufficient for forming droplets (Fig. 5C).
Protein: DP01113 (UniProt D0PV95)
Region: residues 1	to 168
Annotation: RNA binding (Molecular function)
Evidence: fluorescence resonance energy transfer evidence used in manual assertion
Supporting statements:
Because the RGG domain is sufficient for RNA binding (Fig. S8), we asked whether the disordered RGG domain could be responsible for the observed dynamic binding to RNA. Applying our FRET assay to the truncation mutants, we find that both ΔC and the RGG domain alone exhibit highly dynamic FRET traces, similar to that seen in the full-length construct (Fig. 5D). However, ΔRGG gives rise to a static, tightly wrapped conformation of bound RNA, similar to that observed in non–droplet-forming conditions (Fig. 4 B and C).
Protein: DP01114 (UniProt P16333)
Region: residues 59	to 108
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
L1 is partially disordered, and also binds the second SH3 domain.
Protein: DP01114 (UniProt P16333)
Region: residues 59	to 108
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: microscopy evidence used in manual assertion
Supporting statements:
Our studies demonstrate that linker regions between modular domains can contribute to the driving forces for self-assembly and phase separation of multivalent proteins.
Conserved interdomain linker promotes phase separation of the multivalent adaptor protein Nck.
Protein: DP01115 (UniProt Q16236)
Region: residues 69	to 84
Annotation: protein binding (Molecular function)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Keap1 is a BTB-Kelch substrate adaptor protein that regulates steady-state levels of Nrf2, a bZIP transcription factor, in response to oxidative stress. We have determined the structure of the Kelch domain of Keap1 bound to a 16-mer peptide from Nrf2 containing a highly conserved DxETGE motif. The Nrf2 peptide contains two short antiparallel β-strands connected by two overlapping type I β-turns stabilized by the aspartate and threonine residues. The β-turn region fits into a binding pocket on the top face of the Kelch domain and the glutamate residues form multiple hydrogen bonds with highly conserved residues in Keap1.
Cross-references: PDB:2FLU
Protein: DP01115 (UniProt Q16236)
Region: residues 1	to 605
Annotation: pre-molten globule (Structural state)
Evidence: sodium dodecyl sulfate polyacrylamide gel electrophoresis evidence used in manual assertion
Supporting statements:
Intriguingly, although the molecular weight of FL-Nrf2 is only 70.4 kDa (including the His-tag), it runs with an apparent MW of ~110 kDa on SDS-PAGE gels as already reported previously [44] (Figure S1). The high net charge and aberrant SDS-PAGE migration behavior suggest that FL-Nrf2 may be partially disordered
Protein: DP01115 (UniProt Q16236)
Region: residues 1	to 605
Annotation: pre-molten globule (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
CD spectropolarimetry was applied for assessing the secondary structure of FL-Nrf2 at 5, 10, 25, and 35 °C. The negative bands at 208 and 222 nm indicate the presence of α-helical structural elements (Figure 1A). Spectral deconvolution indicates that at 25 °C, there are around 27% α-helical, 21% β-strand and 52% disordered/turn structures, implying that Nrf2 is indeed partially disordered (Figure 1B; Table S1).
Protein: DP01115 (UniProt Q16236)
Region: residues 1	to 605
Annotation: pre-molten globule (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
We used NMR spectroscopy to further verify the disordered nature of FL-Nrf2. Figure 2 shows the 1H-15N HSQC spectra of FL-Nrf2 acquired at 5, 10, 25, and 35 °C. At all four temperatures, despite the presence of some well-dispersed peaks with relatively weak intensities, most of the observed backbone amide signals are crowded in a narrow region between 7.8 and 8.7 ppm in the 1H dimension. This lack of 1H resonance dispersion indicates that many parts of FL-Nrf2 do not adopt stable structures and undergo rapid conformational interconversion [51,52,53,54]. In addition, the disordered nature of FL-Nrf2 does not change significantly at lower temperatures. For comparison, we also acquired the HSQC spectrum of FL-Nrf2 at 35 °C in the presence of 6 M urea, under which conditions the protein was expected to be largely unfolded (Figure S3). The similarity of all these spectra therefore suggest that FL-Nrf2 is already extensively unfolded even in the absence of urea.
Protein: DP01115 (UniProt Q16236)
Region: residues 1	to 605
Annotation: pre-molten globule (Structural state)
Evidence: protein hydrogen-deuterium exchange mass spectrometry evidence used in manual assertion
Supporting statements:
Fast HDX kinetics were observed throughout the entire protein, where the majority of the peptides exhibit ~100% deuteration after 12 s. This lack of protection indicates that FL-Nrf2 is significantly disordered (Figure S6). Notably, several peptides in distinct parts of the protein showed somewhat slower deuteration. For instance, the peptide covering residues 54–74 in the Neh2 domain only displayed complete deuteration after >24 s. This observation is consistent with earlier findings that even though the Neh2 domain is disordered, helical propensity exists between residues 39 and 71 [22]. Other regions that showed somewhat slower exchange are 235–249, 417–434, and 512–537. They are located in the Neh7 domain, the linker between the Neh6 and Neh1 domains, and the Neh1 domain, respectively.
Protein: DP01115 (UniProt Q16236)
Region: residues 17	to 46
Annotation: pre-molten globule to order (Structural transition)
Evidence: protein hydrogen-deuterium exchange mass spectrometry evidence used in manual assertion
Supporting statements:
We also investigated how FL-Nrf2 interacts with Kelch, which is part of the negative regulator Keap1. Previous studies showed that the isolated Neh2 domain of FL-Nrf2 binds Kelch at two sites: the high- affinity ETGE motif (around residues 76–84; Kd ~5 nM) and the low-affinity DLG motif (around residues 17–46; Kd ~1 μM) [22]. Here we used HDX-MS to further dissect the effects of Kelch-binding on the conformational dynamics of FL-Nrf2. In these experiments, the FL-Nrf2 concentration was held constant at 2 μM, while the Kelch concentration varied from 2 to 4 and 6 μM, which corresponded to FL-Nrf2:Kelch ratios of 1:1, 1:2, and 1:3, respectively (Figure 4).
Significant changes in deuterium uptake were observed in the Neh2 domain upon the addition of Kelch. In the presence of 1:1 Kelch, a considerable reduction in FL-Nrf2 deuterium uptake was displayed around the ETGE binding motif and a small reduction in deuterium uptake around the DLG motif, consistent with the binding affinities of the two sites. For a 1:2 ratio, a substantial reduction in deuterium uptake was observed around the DLG binding site and an even more significant reduction in HDX close to the ETGE motif. Further reduction in deuterium uptake around both sites was noted at a 1:3 ratio. Notably, the addition of Kelch did not slow down the deuterium uptake in any other domains of FL-Nrf2.
Protein: DP01115 (UniProt Q16236)
Region: residues 17	to 46
Annotation: protein binding (Molecular function)
Evidence: protein hydrogen-deuterium exchange mass spectrometry evidence used in manual assertion
Supporting statements:
We also investigated how FL-Nrf2 interacts with Kelch, which is part of the negative regulator Keap1. Previous studies showed that the isolated Neh2 domain of FL-Nrf2 binds Kelch at two sites: the high- affinity ETGE motif (around residues 76–84; Kd ~5 nM) and the low-affinity DLG motif (around residues 17–46; Kd ~1 μM) [22]. Here we used HDX-MS to further dissect the effects of Kelch-binding on the conformational dynamics of FL-Nrf2. In these experiments, the FL-Nrf2 concentration was held constant at 2 μM, while the Kelch concentration varied from 2 to 4 and 6 μM, which corresponded to FL-Nrf2:Kelch ratios of 1:1, 1:2, and 1:3, respectively (Figure 4).
Significant changes in deuterium uptake were observed in the Neh2 domain upon the addition of Kelch. In the presence of 1:1 Kelch, a considerable reduction in FL-Nrf2 deuterium uptake was displayed around the ETGE binding motif and a small reduction in deuterium uptake around the DLG motif, consistent with the binding affinities of the two sites. For a 1:2 ratio, a substantial reduction in deuterium uptake was observed around the DLG binding site and an even more significant reduction in HDX close to the ETGE motif. Further reduction in deuterium uptake around both sites was noted at a 1:3 ratio. Notably, the addition of Kelch did not slow down the deuterium uptake in any other domains of FL-Nrf2.
Protein: DP01115 (UniProt Q16236)
Region: residues 76	to 84
Annotation: protein binding (Molecular function)
Evidence: protein hydrogen-deuterium exchange mass spectrometry evidence used in manual assertion
Supporting statements:
We also investigated how FL-Nrf2 interacts with Kelch, which is part of the negative regulator Keap1. Previous studies showed that the isolated Neh2 domain of FL-Nrf2 binds Kelch at two sites: the high- affinity ETGE motif (around residues 76–84; Kd ~5 nM) and the low-affinity DLG motif (around residues 17–46; Kd ~1 μM) [22]. Here we used HDX-MS to further dissect the effects of Kelch-binding on the conformational dynamics of FL-Nrf2. In these experiments, the FL-Nrf2 concentration was held constant at 2 μM, while the Kelch concentration varied from 2 to 4 and 6 μM, which corresponded to FL-Nrf2:Kelch ratios of 1:1, 1:2, and 1:3, respectively (Figure 4).
Significant changes in deuterium uptake were observed in the Neh2 domain upon the addition of Kelch. In the presence of 1:1 Kelch, a considerable reduction in FL-Nrf2 deuterium uptake was displayed around the ETGE binding motif and a small reduction in deuterium uptake around the DLG motif, consistent with the binding affinities of the two sites. For a 1:2 ratio, a substantial reduction in deuterium uptake was observed around the DLG binding site and an even more significant reduction in HDX close to the ETGE motif. Further reduction in deuterium uptake around both sites was noted at a 1:3 ratio. Notably, the addition of Kelch did not slow down the deuterium uptake in any other domains of FL-Nrf2.
Protein: DP01115 (UniProt Q16236)
Region: residues 76	to 84
Annotation: regulation of cellular response to oxidative stress (Biological process)
Evidence: protein hydrogen-deuterium exchange mass spectrometry evidence used in manual assertion
Supporting statements:
Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription regulator that plays a pivotal role in coordinating the cellular response to oxidative stress. Through interactions with other proteins, such as Kelch-like ECH-associated protein 1 (Keap1), CREB-binding protein (CBP), and retinoid X receptor alpha (RXRα), Nrf2 mediates the transcription of cytoprotective genes critical for removing toxicants and preventing DNA damage, thereby playing a significant role in chemoprevention.
Protein: DP01115 (UniProt Q16236)
Region: residues 17	to 46
Annotation: regulation of cellular response to oxidative stress (Biological process)
Evidence: protein hydrogen-deuterium exchange mass spectrometry evidence used in manual assertion
Supporting statements:
Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription regulator that plays a pivotal role in coordinating the cellular response to oxidative stress. Through interactions with other proteins, such as Kelch-like ECH-associated protein 1 (Keap1), CREB-binding protein (CBP), and retinoid X receptor alpha (RXRα), Nrf2 mediates the transcription of cytoprotective genes critical for removing toxicants and preventing DNA damage, thereby playing a significant role in chemoprevention.
Protein: DP01115 (UniProt Q16236)
Region: residues 74	to 87
Annotation: ubiquitin protein ligase binding (Molecular function)
Evidence: qualitative western immunoblotting evidence used in manual assertion
Supporting statements:
Peptide competition experiments were carried out using 14-mer peptides that contained either an alanine or phosphothreonine in place of T80. A 14-mer peptide corresponding to amino acids 74–87 of Nrf2 that contained an alanine in place of T80 was unable to displace HA-Nrf2 from Keap1-CBD even at input peptide levels 100-fold higher than that needed for the wild-type peptide to displace Nrf2 from Keap1 (Figure 7A, compare lanes 3–6 with lanes 7–10). Likewise, a 14-mer peptide that contained a phosphothreonine residue in place of T80 was also unable to displace Nrf2 from Keap1 (Figure 7A, lanes 11–14).
KEAP1 is a substrate-specific adapter of a BCR (BTB-CUL3-RBX1) E3 ubiquitin ligase complex.
Cross-references: ELM:DEG_Kelch_Keap1_1
Protein: DP01115 (UniProt Q16236)
Region: residues 1	to 56
Annotation: ubiquitin protein ligase binding (Molecular function)
Evidence: glutathione S-transferase pull-down assay evidence used in manual assertion
Supporting statements:
We found that wild-type (WT) Neh2(1-56) nicely interacted with Keap1 (WT in Fig. 2B). The D38H and Q43G mutant forms retained the same binding activity as the WT, and the S33R and Q43R mutant forms exhibited slightly stronger binding activity than the WT (Fig. 2C, right panel).
The peptide used in this assessment derives from the mouse form. The Q43R mutant corresponds to the human protein sequence.
KEAP1 is a substrate-specific adapter of a BCR (BTB-CUL3-RBX1) E3 ubiquitin ligase complex.
Protein: DP01115 (UniProt Q16236)
Region: residues 1	to 605
Annotation: non-membrane-bounded organelle (Cellular component)
Evidence: immunofluorescence confocal microscopy evidence used in manual assertion
Supporting statements:
To identify the proteins contained in the biomolecular condensates, IF analysis of mKeap1-mCh and mEGFP-Nrf2 in H1299 cells was performed, and results confirmed that Keap1 and Nrf2 colocalize in the clusters (Fig. 8A).
Fourth, the ANCHOR mutants form p62-dependent biomolecular condensates that are spherical and contain p62, p62 pS351, polyUb, and NRF2; these clusters are not delivered to the autophagy pathway for clearance and are formed by phase separation.
Furthermore, NRF2 was detected in the biomolecular condensates only in the presence of KEAP1 ANCHOR mutants.
Protein: DP01115 (UniProt Q16236)
Region: residues 1	to 208
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
Droplet formation assays revealed that both NRF21-208-mEGFP and NRF2209-605-mEGFP formed phase-separated droplets in a concentration-dependent manner (Fig. 7C). NRF21-208-mEGFP also contains the AD of NRF2 and formed markedly larger droplets than NRF2209-605-mEGFP at the same concentration
Protein: DP01115 (UniProt Q16236)
Region: residues 209	to 605
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
Droplet formation assays revealed that both NRF21-208-mEGFP and NRF2209-605-mEGFP formed phase-separated droplets in a concentration-dependent manner (Fig. 7C).
Protein: DP01115 (UniProt Q16236)
Region: residues 209	to 605
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: fluorescence recovery after photobleaching evidence used in manual assertion
Supporting statements:
Consistent with the FRAP analysis, fusion analysis showed that NRF21-208 droplets fused into one droplet within seconds (Fig. 7E). These results indicated that NRF21-208-mEGFP droplets are dynamic and liquid-like, whereas NRF2209-605-mEGFP droplets had relatively poor dynamics and droplet formation.
Protein: DP01115 (UniProt Q16236)
Region: residues 1	to 208
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: fluorescence recovery after photobleaching evidence used in manual assertion
Supporting statements:
Consistent with the FRAP analysis, fusion analysis showed that NRF21-208 droplets fused into one droplet within seconds (Fig. 7E). These results indicated that NRF21-208-mEGFP droplets are dynamic and liquid-like, whereas NRF2209-605-mEGFP droplets had relatively poor dynamics and droplet formation.
Protein: DP01115 (UniProt Q16236)
Region: residues 1	to 208
Annotation: positive regulation of transcription, DNA-templated (Biological process)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
We expressed either full-length NRF2 fused to EGFP or the NRF2 peptide lacking the first IDR (EGFP-NRF2209-605) in NRF2 knockout cells (fig. S7C). Only full-length NRF2 increases the expression of NRF2 target genes (Fig. 7G), suggesting that the first IDR is required for NRF2 transactivation.
Protein: DP01115 (UniProt Q16236)
Region: residues 1	to 605
Annotation: mediator complex (Cellular component)
Evidence: immunofluorescence evidence used in manual assertion
Supporting statements:
We detected NRF2-positive nuclear puncta in WT 9-12 ADPKD cells, and the size and the number of NRF2-positive puncta increased upon SFN treatment (Fig. 8A). Costaining for NRF2 and MED1 showed that they partially colocalized at puncta in the nuclei of cells (Fig. 8B), suggesting that NRF2 associates with Mediator condensates in vivo.
Together, these results showed that NRF2 and the Mediator complex form phase-separated condensates at superenhancers to regulate NRF2 target gene expression, including cytoprotective genes.
Protein: DP01115 (UniProt Q16236)
Region: residues 1	to 605
Annotation: non-membrane-bounded organelle (Cellular component)
Evidence: immunofluorescence evidence used in manual assertion
Supporting statements:
We detected NRF2-positive nuclear puncta in WT 9-12 ADPKD cells, and the size and the number of NRF2-positive puncta increased upon SFN treatment (Fig. 8A). Costaining for NRF2 and MED1 showed that they partially colocalized at puncta in the nuclei of cells (Fig. 8B), suggesting that NRF2 associates with Mediator condensates in vivo.
Together, these results showed that NRF2 and the Mediator complex form phase-separated condensates at superenhancers to regulate NRF2 target gene expression, including cytoprotective genes.
Protein: DP01116 (UniProt Q03164)
Region: residues 140	to 160
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
In addition, the lack of a defined conformation in the backbone region of bound MLL140–160 between anchored residues F148 and F151 is in agreement with the absence of amide proton signals for G150 and F151 in NMR spectra due to exchange-related signal broadening.
Cross-references: PDB:2MSR
BMRB:25130
Protein: DP01116 (UniProt Q03164)
Region: residues 140	to 160
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
MLL forms a ternary complex with the lens epithelium–derived growth factor (LEDGF/p75) and MENIN.
MLL140–160 is anchored between the two interhelical loops of LEDGF/p75 IBD (aa I359 to D369 and K402 to M413) via F148 and F151 (Fig. 3B).
Cross-references: BMRB:25130
PDB:2MSR
Protein: DP01116 (UniProt Q03164)
Region: residues 140	to 160
Annotation: protein binding (Molecular function)
Evidence: isothermal titration calorimetry evidence used in manual assertion
Supporting statements:
Binding of MLL140–160 to the IBD was further investigated using ITC. The ITC results corresponded with a stoichiometry close to 1:1 (n = 1.15). They were fitted by a single-site binding model, which provided the dissociation constant (Kd) of 86.3 ± 21.3 μmol/L (Fig. 2E). These results confirmed the existence of an additional interface between LEDGF/p75 and MLL.
Protein: DP01117 (UniProt O60566)
Region: residues 20	to 42
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The region represents a linear degradation motif [degron]: DEG_APCC_KENBOX_2
Cross-references: PDB:4GGD
ELM:DEG_APCC_KENBOX_2
ELM:ELMI002601
Protein: DP01117 (UniProt O60566)
Region: residues 20	to 42
Annotation: protein binding (Molecular function)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Checkpoint inhibition of APC/CCdc20 requires the binding of a BubR1 KEN box to Cdc20.
Cross-references: ELM:DEG_APCC_KENBOX_2
ELM:ELMI002601
PDB:4GGD
Protein: DP01117 (UniProt O60566)
Region: residues 20	to 42
Annotation: protein binding (Molecular function)
Evidence: radioisotope assay evidence used in manual assertion
Supporting statements:
Addition of the synthetic BubR1 KEN1 peptide reduced, but did not abolish, Cdc20 binding to BubR1N, indicating that other regions of BubR1N contributed to Cdc20 binding (Fig. S2B).
Cross-references: ELM:DEG_APCC_KENBOX_2
Protein: DP01118 (UniProt Q12834)
Region: residues 81	to 164
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Both the N-terminal region (residues 81–164) and the C-terminal region (residues 477–499; including the IR motif) of Cdc20ΔN80 were disordered and not visible in the structure.
Cross-references: PDB:4GGD
Protein: DP01118 (UniProt Q12834)
Region: residues 477	to 499
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Both the N-terminal region (residues 81–164) and the C-terminal region (residues 477–499; including the IR motif) of Cdc20ΔN80 were disordered and not visible in the structure.
Cross-references: PDB:4GGD
Protein: DP01119 (UniProt P35222)
Region: residues 19	to 44
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
In the case of the β-catenin substrate, the segment between the destruction motif and the lysine is unstructured
Cross-references: PDB:1P22
Protein: DP01119 (UniProt P35222)
Region: residues 692	to 781
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Only residues 665–691 of β-catenin C-terminal domain, which were also identified by MR method, were visible in the electron density maps before and after density modification.
Cross-references: PDB:2Z6H
Protein: DP01119 (UniProt P35222)
Region: residues 687	to 781
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The 15N-TROSY spectrum of 15N-labeled β-catenin C-terminal tail alone shows poor dispersion of the resonance, indicating that the C-terminal tail per se does not have any folded structure.
Protein: DP01119 (UniProt P35222)
Region: residues 1	to 137
Annotation: disorder (Structural state)
Evidence: cleavage assay evidence used in manual assertion
Supporting statements:
The N-terminal domain of β-catenin has a poor expression yield and tends to be degraded during purification.