LiteFold/DisProt huggingface dataset:
Protein: DP00959 (UniProt O15169)
Region: residues 295	to 500
Annotation: molecular adaptor activity (Molecular function)
Evidence: radioisotope assay evidence used in manual assertion
Supporting statements:
Titration of AxinCRG295-A500 strongly enhanced the efficiency of GSK3β in phosphorylating β-catenin in vitro (Fig. 8a), whereas no effect was observed on AxinCR variants lacking either the β-catenin binding site (AxinCRΔβcat) or the GSK3β binding site (AxinCRΔGSK3β), suggesting that Axin binding of both GSK3β and β-catenin is required for its scaffolding function (Fig. 8b and c).
We conclude that the highly flexible AxinCRG295-A500 fragment scaffolds and targets GSK3β kinase activity towards its substrate, β-catenin.
Protein: DP00959 (UniProt O15169)
Region: residues 295	to 500
Annotation: beta-catenin binding (Molecular function)
Evidence: glutathione S-transferase pull-down assay evidence used in manual assertion
Supporting statements:
We recombinantly produced GSK3β as His-tagged NusA fusion protein (to increase solubility) and His-tagged β-catenin. Both His-NusA-tagged GSK3β and His-tagged β-catenin, but not control His-NusA protein, were able to bind AxinCRG295-A500 in a pull-down approach (Fig. 2a–c).
To verify that this interaction also takes place in vivo, we expressed glutathione S-transferase (GST)-tagged AxinCRG295-A500 in HEK293T cells and confirmed its binding to endogenous GSK3β and β-catenin (Fig. 2d).
Protein: DP00959 (UniProt O15169)
Region: residues 295	to 500
Annotation: protein binding (Molecular function)
Evidence: glutathione S-transferase pull-down assay evidence used in manual assertion
Supporting statements:
We recombinantly produced GSK3β as His-tagged NusA fusion protein (to increase solubility) and His-tagged β-catenin. Both His-NusA-tagged GSK3β and His-tagged β-catenin, but not control His-NusA protein, were able to bind AxinCRG295-A500 in a pull-down approach (Fig. 2a–c).
To verify that this interaction also takes place in vivo, we expressed glutathione S-transferase (GST)-tagged AxinCRG295-A500 in HEK293T cells and confirmed its binding to endogenous GSK3β and β-catenin (Fig. 2d).
Protein: DP00963 (UniProt P0DJ89)
Region: residues 293	to 314
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
In contrast, the C-terminal part (residues 17–47), which includes the proline-rich segment 27IPPAPNWPAPTPP39 that harbors the tandem PxxP motifs responsible IRTKS SH3 binding, is highly disordered.
Protein: DP00963 (UniProt P0DJ89)
Region: residues 294	to 306
Annotation: disorder to order (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Clearly, the N-WASP GBD binding epitope, encompassing residues 3Asp-Arg21, became more rigid upon binding to N-WASP as reported by increased heteronuclear NOEs > 0.7), whereas extensive disorder-to-order transition, which translates into substantial increase of heteronuclear NOEs from negative to large positive values, could be observed for the IRTKS SH3 binding region (27IPPAPNWPAPTPP39).
 It contains two protein recognition motifs, which undergo disorder-to-order transition upon binding.
Cross-references: BMRB:18165
PDB:2LNH
Protein: DP00963 (UniProt P0DJ89)
Region: residues 294	to 306
Annotation: SH3 domain binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Clearly, the N-WASP GBD binding epitope, encompassing residues 3Asp-Arg21, became more rigid upon binding to N-WASP as reported by increased heteronuclear NOEs > 0.7), whereas extensive disorder-to-order transition, which translates into substantial increase of heteronuclear NOEs from negative to large positive values, could be observed for the IRTKS SH3 binding region (27IPPAPNWPAPTPP39)
Cross-references: BMRB:18165
PDB:2LNH
Protein: DP00963 (UniProt P0DJ89)
Region: residues 268	to 314
Annotation: disorder (Structural state)
Evidence: heteronuclear single quantum coherence spectroscopy evidence used in manual assertion
Supporting statements:
Instead of well-dispersed 15N-1H correlation spectrum of folded proteins, the 15N-HSQC spectrum of EspFU R475 displayed a poorly dispersed correlation map reminiscent of disordered polypeptide chain (Fig. 1C).
Protein: DP00963 (UniProt P0DJ89)
Region: residues 294	to 306
Annotation: exogenous protein binding (Molecular function)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
W to A replacement in EspFU peptide reduced the affinity substantially, Kd (EspFUW33A) = 22.3 μM, and strikingly, the A to W replacement converted the Eps8 peptide into a strong binder Kd (Eps8A33W) = 2.4 μM. This clearly pinpointed the critical role of the linker tryptophan for high affinity, thus explaining the higher affinity of EspFU as compared to the cellular Eps8 ligand.
Next, we tested alanine substitution of residue W33 in a yeast two-hybrid assay, and found that W to A mutation in “PW33A” construct disrupted binding to IRTKS-SH3, and completely abrogated any activity in this assay (Fig. 4C).
Protein: DP00963 (UniProt P0DJ89)
Region: residues 294	to 306
Annotation: adhesion of symbiont to host cell (Biological process)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
When MEFs expressing wild type GFP-HPHP fusion construct were infected with KC12, a modified E. coli capable of translocating Tir but not expressing EspFU, the ectopically expressed GFP-HPHP was recruited to the sites of bacterial attachment, as shown by immunostaining the myc-tagged GFP-HPHP fusion, and induced actin pedestal formation (Fig. 4D). In contrast, the IRTKS binding-deficient mutant GFP-HP*HP* failed to be recruited to the sites of bacterial attachments, and consequently no actin assembly was observed, suggesting that IRTKS binding was an essential activity for EspFU recruitment to sites of clustered Tir.
Thus, we conclude that the enhanced IRTKS binding affinity provided by W33 plays a critical role in pedestal formation.
Protein: DP00963 (UniProt P0DJ89)
Region: residues 294	to 306
Annotation: positive regulation of actin nucleation (Biological process)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
When MEFs expressing wild type GFP-HPHP fusion construct were infected with KC12, a modified E. coli capable of translocating Tir but not expressing EspFU, the ectopically expressed GFP-HPHP was recruited to the sites of bacterial attachment, as shown by immunostaining the myc-tagged GFP-HPHP fusion, and induced actin pedestal formation (Fig. 4D). In contrast, the IRTKS binding-deficient mutant GFP-HP*HP* failed to be recruited to the sites of bacterial attachments, and consequently no actin assembly was observed, suggesting that IRTKS binding was an essential activity for EspFU recruitment to sites of clustered Tir.
Thus, we conclude that the enhanced IRTKS binding affinity provided by W33 plays a critical role in pedestal formation.
Protein: DP00963 (UniProt P0DJ89)
Region: residues 268	to 314
Annotation: modulation by symbiont of host process (Biological process)
Evidence: combinatorial experimental and curator inference evidence used in manual assertion
Supporting statements:
Most importantly, we reveal the underlying structural mechanism by which EspFU outcompetes cellular IRTKS SH3 binding ligands and firmly interconnects actin polymerization and membrane regulation machineries.
These data showed that the pathogenic EspFU is able to usurp this host cell signaling pathway by superseding the cellular ligand with approximately 60 times higher affinity against its target.
When MEFs expressing wild type GFP-HPHP fusion construct were infected with KC12, a modified E. coli capable of translocating Tir but not expressing EspFU, the ectopically expressed GFP-HPHP was recruited to the sites of bacterial attachment, as shown by immunostaining the myc-tagged GFP-HPHP fusion, and induced actin pedestal formation (Fig. 4D).
Thus, we conclude that the enhanced IRTKS binding affinity provided by W33 plays a critical role in pedestal formation.
Protein: DP00963 (UniProt P0DJ89)
Region: residues 268	to 314
Annotation: molecular adaptor activity (Molecular function)
Evidence: heteronuclear single quantum coherence spectroscopy evidence used in manual assertion
Supporting statements:
Despite subtle chemical shift perturbations observed in 15N-HSQC spectra of EspFU R475 between binary and ternary complexes (Fig. 2D), the structure suggests that the N- and C-terminal regions of EspFU function as independent units.
Thus, EspFU binding of the IRTKS/IRSp53 SH3 domain results in recruitment of the EspFU:N-WASP:Arp2/3 complex and localized actin assembly.
Cross-references: BMRB:18165
PDB:2LNH
Protein: DP00963 (UniProt P0DJ89)
Region: residues 270	to 288
Annotation: disorder to order (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Clearly, the N-WASP GBD binding epitope, encompassing residues 3Asp-Arg21, became more rigid upon binding to N-WASP as reported by increased heteronuclear NOEs > 0.7), whereas extensive disorder-to-order transition, which translates into substantial increase of heteronuclear NOEs from negative to large positive values, could be observed for the IRTKS SH3 binding region (27IPPAPNWPAPTPP39).
 It contains two protein recognition motifs, which undergo disorder-to-order transition upon binding.
Cross-references: BMRB:18165 
PDB:2LNH
Protein: DP00963 (UniProt P0DJ89)
Region: residues 270	to 288
Annotation: exogenous protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Clearly, the N-WASP GBD binding epitope, encompassing residues 3Asp-Arg21, became more rigid upon binding to N-WASP as reported by increased heteronuclear NOEs > 0.7), whereas extensive disorder-to-order transition, which translates into substantial increase of heteronuclear NOEs from negative to large positive values, could be observed for the IRTKS SH3 binding region (27IPPAPNWPAPTPP39).
Cross-references: BMRB:18165 
PDB:2LNH
Protein: DP00963 (UniProt P0DJ89)
Region: residues 221	to 267
Annotation: disorder (Structural state)
Evidence: heteronuclear single quantum coherence spectroscopy evidence used in manual assertion
Supporting statements:
Instead of well-dispersed 15N-1H correlation spectrum of folded proteins, the 15N-HSQC spectrum of EspFU R475 displayed a poorly dispersed correlation map reminiscent of disordered polypeptide chain (Fig. 1C).
Protein: DP00963 (UniProt P0DJ89)
Region: residues 246	to 267
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
In contrast, the C-terminal part (residues 17–47), which includes the proline-rich segment 27IPPAPNWPAPTPP39 that harbors the tandem PxxP motifs responsible IRTKS SH3 binding, is highly disordered.
Protein: DP00963 (UniProt P0DJ89)
Region: residues 223	to 241
Annotation: exogenous protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Clearly, the N-WASP GBD binding epitope, encompassing residues 3Asp-Arg21, became more rigid upon binding to N-WASP as reported by increased heteronuclear NOEs > 0.7), whereas extensive disorder-to-order transition, which translates into substantial increase of heteronuclear NOEs from negative to large positive values, could be observed for the IRTKS SH3 binding region (27IPPAPNWPAPTPP39).
Cross-references: BMRB:18165
PDB:2LNH
Protein: DP00963 (UniProt P0DJ89)
Region: residues 247	to 259
Annotation: exogenous protein binding (Molecular function)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
W to A replacement in EspFU peptide reduced the affinity substantially, Kd (EspFUW33A) = 22.3 μM, and strikingly, the A to W replacement converted the Eps8 peptide into a strong binder Kd (Eps8A33W) = 2.4 μM. This clearly pinpointed the critical role of the linker tryptophan for high affinity, thus explaining the higher affinity of EspFU as compared to the cellular Eps8 ligand.
Next, we tested alanine substitution of residue W33 in a yeast two-hybrid assay, and found that W to A mutation in “PW33A” construct disrupted binding to IRTKS-SH3, and completely abrogated any activity in this assay (Fig. 4C).
Protein: DP00963 (UniProt P0DJ89)
Region: residues 223	to 241
Annotation: disorder to order (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Clearly, the N-WASP GBD binding epitope, encompassing residues 3Asp-Arg21, became more rigid upon binding to N-WASP as reported by increased heteronuclear NOEs > 0.7), whereas extensive disorder-to-order transition, which translates into substantial increase of heteronuclear NOEs from negative to large positive values, could be observed for the IRTKS SH3 binding region (27IPPAPNWPAPTPP39).
It contains two protein recognition motifs, which undergo disorder-to-order transition upon binding.
Cross-references: BMRB:18165
PDB:2LNH
Protein: DP00963 (UniProt P0DJ89)
Region: residues 247	to 259
Annotation: disorder to order (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Clearly, the N-WASP GBD binding epitope, encompassing residues 3Asp-Arg21, became more rigid upon binding to N-WASP as reported by increased heteronuclear NOEs > 0.7), whereas extensive disorder-to-order transition, which translates into substantial increase of heteronuclear NOEs from negative to large positive values, could be observed for the IRTKS SH3 binding region (27IPPAPNWPAPTPP39).
It contains two protein recognition motifs, which undergo disorder-to-order transition upon binding.
Cross-references: BMRB:18165
PDB:2LNH
Protein: DP00964 (UniProt P60460)
Region: residues 1	to 20
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The PDB structure of the SecYEβ complexes shows this region lacks electron density, indicating it is disordered.
Cross-references: PDB:1RH5
PDB:1RHZ
Protein: DP00964 (UniProt P60460)
Region: residues 1	to 20
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The PDB structure of the SecYEβ complexes shows this region lacks electron density, indicating it is disordered.
Cross-references: PDB:2YXQ
PDB:2YXR
Protein: DP00969 (UniProt Q02548)
Region: residues 1	to 30
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
However, the N-terminal ~ 30 residues of Pax51-92 and Pax51-149 lacked any persistent secondary structure and exhibited low RCI-S2 values, indicative of conformational flexibility.
Cross-references: BMRB:26730
Protein: DP00969 (UniProt Q02548)
Region: residues 19	to 30
Annotation: disorder to order (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy-based hydrogen-deuterium exchange evidence used in manual assertion
Supporting statements:
In contrast, the RCI-S2 values of the linker and the N-terminal residues increased to match those in the helical bundles. This indicates that the protein backbone in these regions becomes more ordered when bound to DNA (Fig. 3b).
Cross-references: BMRB:26730
BMRB:26731
Protein: DP00969 (UniProt Q02548)
Region: residues 76	to 92
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy-based hydrogen-deuterium exchange evidence used in manual assertion
Supporting statements:
The linker residues in all three fragments were also found to be disordered, exhibiting random coil chemical shifts and hence low RCI-S2 parameters.
Cross-references: BMRB:26730
Protein: DP00969 (UniProt Q02548)
Region: residues 76	to 92
Annotation: flexible linker (Disorder function)
Evidence: nuclear magnetic resonance spectroscopy-based hydrogen-deuterium exchange evidence used in manual assertion
Supporting statements:
The linker residues in all three fragments were also found to be disordered, exhibiting random coil chemical shifts and hence low RCI-S2 parameters.
Protein: DP00969 (UniProt Q02548)
Region: residues 76	to 92
Annotation: disorder to order (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy-based hydrogen-deuterium exchange evidence used in manual assertion
Supporting statements:
In contrast, the RCI-S2 values of the linker and the N-terminal residues increased to match those in the helical bundles. This indicates that the protein backbone in these regions becomes more ordered when bound to DNA (Fig. 3b).
Cross-references: BMRB:26730
BMRB:26731
Protein: DP00969 (UniProt Q02548)
Region: residues 1	to 18
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The final model had all the DNA, Pax-5 residues 19–142, and Ets-1 residues 309–437.
Cross-references: PDB:1MDM
Protein: DP00973 (UniProt P46108-2)
Region: residues 121	to 133
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
CRKI (residues 1–204) showed well-dispersed, sharp signals, which we did not expect, given its molecular weight. Thus, CRKI was considered to have well-defined SH2 and SH3 domains that were connected by a flexible linker.
Cross-references: PDB:2EYY
Protein: DP00973 (UniProt P46108-2)
Region: residues 121	to 133
Annotation: flexible linker (Disorder function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
CRKI (residues 1–204) showed well-dispersed, sharp signals, which we did not expect, given its molecular weight. Thus, CRKI was considered to have well-defined SH2 and SH3 domains that were connected by a flexible linker.
Cross-references: PDB:2EYY
Protein: DP00974 (UniProt P42866)
Region: residues 1	to 51
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Previously crystallized GPCRs show little density for the poorly ordered amino and carboxy terminal domains. Although these domains are not critical for maintaining high ligand affinity, these flexible regions may inhibit crystallogenesis
Cross-references: PDB:4DKL
Protein: DP00974 (UniProt P42866)
Region: residues 361	to 398
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Previously crystallized GPCRs show little density for the poorly ordered amino and carboxy terminal domains. Although these domains are not critical for maintaining high ligand affinity, these flexible regions may inhibit crystallogenesis
Cross-references: PDB:4DKL
Protein: DP00975 (UniProt Q86FP9)
Region: residues 50	to 73
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The electron density maps were readily interpretable for madanin-1 residues M D31 to M R54 (Fig. 5a)...
Cross-references: PDB:5L6N
Protein: DP00975 (UniProt Q86FP9)
Region: residues 50	to 73
Annotation: molecular function inhibitor activity (Molecular function)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Madanin-1 exhibits a novel mechanism of thrombin inhibition.
Protein: DP00975 (UniProt Q86FP9)
Region: residues 50	to 73
Annotation: protein binding (Molecular function)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Remarkably, madanin-1 runs along the surface of thrombin in an extended conformation, with its C-terminal region blocking the active site of the enzyme, while the sulfotyrosine-containing region of the inhibitor establishes specific interactions with the exosite II of thrombin (Fig. 5a).
Protein: DP00975 (UniProt Q86FP9)
Region: residues 50	to 73
Annotation: disorder to order (Structural transition)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Remarkably, madanin-1 runs along the surface of thrombin in an extended conformation, with its C-terminal region blocking the active site of the enzyme, while the sulfotyrosine-containing region of the inhibitor establishes specific interactions with the exosite II of thrombin (Fig. 5a).
Protein: DP00976 (UniProt P04578)
Region: residues 127	to 196
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with high relative B-factor values used in manual assertion
Supporting statements:
In addition, several of the biologically important loops displayed high atomic-mobility (B) factors and were not accurately modeled.
The region from 127 to 196 (V1-V2 loops) was not modeled.
Protein: DP00976 (UniProt P04578)
Region: residues 127	to 196
Annotation: protein binding (Molecular function)
Evidence: X-ray crystallography-based structural model with high relative B-factor values used in manual assertion
Supporting statements:
For the bridging sheet, the flexibility in the V1/V2 stem in conjunction with a lattice contact with the second domain of CD4 results in a different orientation of the V1/V2 stem and a high overall rmsd.
Protein: DP00976 (UniProt P04578)
Region: residues 127	to 196
Annotation: evasion of host immune response (Biological process)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
The structure of the HIV gp120 envelope glycoprotein allows direct visualization of two overlapping sets of machinery. One, involved in entry, binds to both CD4 and a chemokine receptor and transmits a signal to gp41 to initiate membrane fusion. The other uses loop variation, steric occlusion, conformational change and a carbohydrate cloak to evade the immune system.
Protein: DP00976 (UniProt P04578)
Region: residues 398	to 409
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
These include a single addition to the mostly disordered V4 loop (at 397) and four additional N-acetylglucosamine glycans at asparagines 88, 230, 241, and 463.
Still others, such as the V4 loop, are divergent in both sequence and structure.
Cross-references: PDB:1G9M
Protein: DP00976 (UniProt P04578)
Region: residues 127	to 196
Annotation: viral entry into host cell (Biological process)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
The structure of the HIV gp120 envelope glycoprotein allows direct visualization of two overlapping sets of machinery. One, involved in entry, binds to both CD4 and a chemokine receptor and transmits a signal to gp41 to initiate membrane fusion. The other uses loop variation, steric occlusion, conformational change and a carbohydrate cloak to evade the immune system.
For the bridging sheet, the flexibility in the V1/V2 stem in conjunction with a lattice contact with the second domain of CD4 results in a different orientation of the V1/V2 stem and a high overall rmsd.
Protein: DP00980 (UniProt P36595)
Region: residues 1	to 59
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The N-terminal of the sequence is missing from the pdb, even if not mention in the paper.
Cross-references: PDB:3H0G