LiteFold/DisProt huggingface dataset:
Protein: DP01752 (UniProt Q60598)
Region: residues 83	to 324
Annotation: disorder (Structural state)
Evidence: small-angle X-ray scattering evidence used in manual assertion
Supporting statements:
The repeat region of cortactin is intrinsically disordered in solution
Using circular dichroism (CD) we find that in solution the cortactin repeats resemble a coil-like intrinsically disordered protein. Small-angle X-ray scattering (SAXS) also indicates that the cortactin repeats are intrinsically unfolded, and the experimentally observed radius of gyration (R g) is coincidental to that calculated by the program Flexible-Meccano for an unfolded peptide of this length.
Small-angle X-ray scattering finds the cortactin repeats to be intrinsically disordered
We found no aggregation, and molecular weight estimation based on Porod volume corresponded well with those expected for a monomeric protein (Table 1), however Guinier approximations indicated a radius of gyration (R g) for this 324 amino acid protein of ~47.5 Å (Fig. 3B, Table 1), significantly larger than would be expected for a globular protein (~20 Å)29. Analysis of the scattering properties of cortactinCRH shows that it displays other features expected for intrinsically disordered proteins: its Kratky plot displays a monotonic increase characteristic of intrinsic disorder24 (Fig. 3C), and its Porod-Debye plot does not plateau as would be expected for a globular protein30 (Fig. 3D).
Protein: DP01752 (UniProt Q60598)
Region: residues 83	to 324
Annotation: disorder (Structural state)
Evidence: protein hydrogen-deuterium exchange mass spectrometry evidence used in manual assertion
Supporting statements:
The repeat region of cortactin is intrinsically disordered in solution
Using circular dichroism (CD) we find that in solution the cortactin repeats resemble a coil-like intrinsically disordered protein. Small-angle X-ray scattering (SAXS) also indicates that the cortactin repeats are intrinsically unfolded, and the experimentally observed radius of gyration (R g) is coincidental to that calculated by the program Flexible-Meccano for an unfolded peptide of this length.
Hydrogen-deuterium exchange mass spectrometry shows rapid exchange for most regions of the cortactin repeats
We find most regions of cortactinCR rapidly reached HDX saturation by the first time-point (Fig. 5), indicating that cortactinCR contains minimal hydrophobic core (unprotected) and is largely intrinsically disordered.
Protein: DP01754 (UniProt Q8BHZ5)
Region: residues 1	to 157
Annotation: disorder (Structural state)
Evidence: protein hydrogen-deuterium exchange mass spectrometry evidence used in manual assertion
Supporting statements:
Many of these peptides displayed near maximal deuterium uptake at the earliest time point (10 seconds) suggesting that MRI is intrinsically disordered in solution (Fig. S7C).
Protein: DP01754 (UniProt Q8BHZ5)
Region: residues 1	to 157
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Indeed, while circular dichroism (CD) spectroscopy also demonstrates that MRI is an intrinsically disordered protein, the addition of trifluoroethanol (TFE), a helix-inducing crowding agent, resulted in CD spectra suggestive that MRI has the potential to form helical structures (Fig. S7D) (Lopes et al., 2014).
Protein: DP01754 (UniProt Q8BHZ5)
Region: residues 1	to 157
Annotation: protein homooligomerization (Biological process)
Evidence: static light scattering assay evidence used in manual assertion
Supporting statements:
Finally, size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) revealed that, in solution, MRI can exist as a monomer, dimer, or multimer (Fig. S7E).
Protein: DP01754 (UniProt Q8BHZ5)
Region: residues 1	to 157
Annotation: protein binding (Molecular function)
Evidence: co-immunoprecipitation evidence used in manual assertion
Supporting statements:
Immunoprecipitation with the 13E10.E12.C10 anti-MRI monoclonal antibody confirmed that endogenous MRI associates with DNA-PKcs, ATM, Ku70, Mre11, Nbs1, Rad50, XLF and PAXX (Fig. 4D).
Protein: DP01754 (UniProt Q8BHZ5)
Region: residues 1	to 157
Annotation: DNA repair complex (Cellular component)
Evidence: co-immunoprecipitation evidence used in manual assertion
Supporting statements:
Immunoprecipitation with the 13E10.E12.C10 anti-MRI monoclonal antibody confirmed that endogenous MRI associates with DNA-PKcs, ATM, Ku70, Mre11, Nbs1, Rad50, XLF and PAXX (Fig. 4D).
Protein: DP01754 (UniProt Q8BHZ5)
Region: residues 1	to 17
Annotation: protein binding (Molecular function)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
FLAG-HA-tagged MRI, MRIΔC, MRIΔN or MRIΔNΔC were expressed in WT abl pre-B cells and immunoprecipitated with anti-HA. Members of the DNA-PK complex – Ku70, Ku80 and DNA-PKcs – as well as XLF, PAXX, and XRCC4 all interact with MRI and MRIΔC, but do not interact with MRIΔN or MRIΔNΔC, indicating that the association of these proteins with MRI depends on the N-terminal KBM (Fig. 4E).
Protein: DP01754 (UniProt Q8BHZ5)
Region: residues 143	to 157
Annotation: protein binding (Molecular function)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
Conversely, KAP-1, ATM, and components of the MRN complex - Mre11, Nbs1 and Rad50 - all interact with MRI and MRIΔN, but not MRIΔC or MRIΔNΔC, indicating that the binding of these proteins to MRI depends on the C-terminal XLM (Fig. 4E).
Protein: DP01754 (UniProt Q8BHZ5)
Region: residues 1	to 157
Annotation: molecular adaptor activity (Molecular function)
Evidence: gel-filtration evidence used in manual assertion
Supporting statements:
Third, size-exclusion chromatography revealed that, in abl pre-B cells, MRI exists in fractions that range in size from 100 kDa to over 1 MDa (Fig. 5D). The 30 kDa MRI protein was most abundant in 200 kDa fractions that contained Ku and in fractions greater than 1 MDa that contained Ku, Mre11, ATM, and DNA-PKcs (Fig. 5D). We conclude that MRI is an adaptor that forms large multimeric complexes containing DDR proteins bound at the N-terminus, C-terminus, or both.
Protein: DP01754 (UniProt Q8BHZ5)
Region: residues 1	to 157
Annotation: positive regulation of double-strand break repair via nonhomologous end joining (Biological process)
Evidence: mutant physiological response evidence used in manual assertion
Supporting statements:
Several lines of evidence demonstrate that MRI promotes cNHEJ. MRI is required for cNHEJ-dependent repair of RAG DSBs in XLF-deficient G1-phase abl pre-B cells and for cNHEJ-mediated repair of I-PpoI DSBs in G1- and G2-phase XLF-deficient cells (Fig. 3, S4, and S5).
Protein: DP01754 (UniProt Q8BHZ5)
Region: residues 1	to 157
Annotation: protein localization to site of double-strand break (Biological process)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
Robust MRI-GFP localization was also observed upon laser-induced DNA damage of serum starved G1-phase MRI−/− MEFs (Fig. S2D). We conclude that, like the core cNHEJ factor Ku, MRI localizes to DNA damage sites in MEFs, and MRI deficiency in MEFs leads to IR sensitivity, a common hallmark of cNHEJ deficiency.
Protein: DP01754 (UniProt Q8BHZ5)
Region: residues 1	to 157
Annotation: double-strand break repair via nonhomologous end joining (Biological process)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
Robust MRI-GFP localization was also observed upon laser-induced DNA damage of serum starved G1-phase MRI−/− MEFs (Fig. S2D). We conclude that, like the core cNHEJ factor Ku, MRI localizes to DNA damage sites in MEFs, and MRI deficiency in MEFs leads to IR sensitivity, a common hallmark of cNHEJ deficiency.
Protein: DP01754 (UniProt Q8BHZ5)
Region: residues 1	to 157
Annotation: damaged DNA binding (Molecular function)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
MRI-GFP remained associated with DNA damage sites for at least 45 minutes (Fig. S2C).
Robust MRI-GFP localization was also observed upon laser-induced DNA damage of serum starved G1-phase MRI−/− MEFs (Fig. S2D). We conclude that, like the core cNHEJ factor Ku, MRI localizes to DNA damage sites in MEFs, and MRI deficiency in MEFs leads to IR sensitivity, a common hallmark of cNHEJ deficiency.
Protein: DP01754 (UniProt Q8BHZ5)
Region: residues 1	to 157
Annotation: cellular response to DNA damage stimulus (Biological process)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
MRI-GFP remained associated with DNA damage sites for at least 45 minutes (Fig. S2C).
Robust MRI-GFP localization was also observed upon laser-induced DNA damage of serum starved G1-phase MRI−/− MEFs (Fig. S2D). We conclude that, like the core cNHEJ factor Ku, MRI localizes to DNA damage sites in MEFs, and MRI deficiency in MEFs leads to IR sensitivity, a common hallmark of cNHEJ deficiency.
Protein: DP01757 (UniProt Q13029)
Region: residues 197	to 341
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Signals in NMR spectra of AR displayed narrow line widths and limited dispersion typical of unfolded proteins (Figure 3), and the absence of residues with positive heteronuclear 1H–15N NOE indicated that AR is highly flexible (Figure 4).
Protein: DP01757 (UniProt Q13029)
Region: residues 197	to 341
Annotation: disorder (Structural state)
Evidence: gel-filtration evidence used in manual assertion
Supporting statements:
AR eluted from a Superdex 75 size-exclusion column in a volume similar to that of the 60 kDa bovine albumin standard (data not shown), suggesting that it either formed oligomers or adopted an extended conformation in solution.
Protein: DP01757 (UniProt Q13029)
Region: residues 197	to 269
Annotation: disorder (Structural state)
Evidence: heteronuclear single quantum coherence spectroscopy evidence used in manual assertion
Supporting statements:
The 2D 1H–15N HSQC spectra of AR-N and AR-C are shown in Figure 9. For virtually every signal in these spectra, there is a counterpart at the same position in the 1H–15N HSQC spectrum of AR (data not shown).
Protein: DP01757 (UniProt Q13029)
Region: residues 255	to 267
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Residues 306–322 (KEPEIRCDEKPEDLLEE) were the most affected by RbAB binding (Figure 7). This sequence contains the IRCDE motif that was proposed to mediate the interaction with Rb.21 Surprisingly, residues 255–267 (RLEAAACEVNDLG) were also affected, but the decrease in their signal intensities was delayed relative to that of residues 306–322 (Figure 7). This suggested that the pocket domain binds preferentially to residues 306–322 and that it interacts with residues 255–267 with a lower affinity.
Protein: DP01757 (UniProt Q13029)
Region: residues 297	to 341
Annotation: disorder (Structural state)
Evidence: heteronuclear single quantum coherence spectroscopy evidence used in manual assertion
Supporting statements:
The 2D 1H–15N HSQC spectra of AR-N and AR-C are shown in Figure 9. For virtually every signal in these spectra, there is a counterpart at the same position in the 1H–15N HSQC spectrum of AR (data not shown).
Protein: DP01757 (UniProt Q13029)
Region: residues 306	to 322
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Residues 306–322 (KEPEIRCDEKPEDLLEE) were the most affected by RbAB binding (Figure 7). This sequence contains the IRCDE motif that was proposed to mediate the interaction with Rb.21 Surprisingly, residues 255–267 (RLEAAACEVNDLG) were also affected, but the decrease in their signal intensities was delayed relative to that of residues 306–322 (Figure 7). This suggested that the pocket domain binds preferentially to residues 306–322 and that it interacts with residues 255–267 with a lower affinity.
Protein: DP01757 (UniProt Q13029)
Region: residues 297	to 341
Annotation: protein binding (Molecular function)
Evidence: isothermal titration calorimetry evidence used in manual assertion
Supporting statements:
AR-C bound to RbAB with a Kd of 640 nM, but no interaction between AR-N and RbAB was detected (Figure 8B,C and Table 1).
Protein: DP01757 (UniProt Q13029)
Region: residues 254	to 267
Annotation: protein binding (Molecular function)
Evidence: fluorescence anisotropy evidence used in manual assertion
Supporting statements:
The interaction of FITC-RIZ(254–267) with RbAB was readily observed by this method (Figure 11A), and fitting of the anisotropy data yielded a Kd of ~170 μM (Table 2).
Protein: DP01757 (UniProt Q13029)
Region: residues 254	to 267
Annotation: protein binding (Molecular function)
Evidence: competitive binding evidence used in manual assertion
Supporting statements:
We also used a competitive binding assay with the FITC-RIZ(254–267) peptide (Figure 11B) and established that AR-N binds to RbAB with comparable affinity (Kd ~ 90 μM) (Table 2).
Protein: DP01757 (UniProt Q13029)
Region: residues 309	to 319
Annotation: protein binding (Molecular function)
Evidence: competitive binding evidence used in manual assertion
Supporting statements:
Fluorescence anisotropy experiments with the FITC-RIZ(309–319) peptide, which contains the IRCDE motif, and competition assays with AR-C (Figure 11C,D) yielded Kd values of ~360 and ~110 nM for the interaction of RbAB with the FITC-RIZ(309–319) peptide and AR-C, respectively (Table 2).
Protein: DP01757 (UniProt Q13029)
Region: residues 309	to 319
Annotation: protein binding (Molecular function)
Evidence: fluorescence anisotropy evidence used in manual assertion
Supporting statements:
Fluorescence anisotropy experiments with the FITC-RIZ(309–319) peptide, which contains the IRCDE motif, and competition assays with AR-C (Figure 11C,D) yielded Kd values of ~360 and ~110 nM for the interaction of RbAB with the FITC-RIZ(309–319) peptide and AR-C, respectively (Table 2).
Protein: DP01759 (UniProt Q0GBY3)
Region: residues 1	to 68
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The far-UV CD spectrum of full-length P, PCED, PCTD, and PΔ91–131 exhibited two dichroic bands near 208 nm and 222 nm, indicating the presence of α-helical structure, while the spectrum obtained with PNTD showed one minor dichroic band near 222 nm and one below 200 nm, suggesting a low α-helical content (Fig. 6).
Protein: DP01759 (UniProt Q0GBY3)
Region: residues 1	to 68
Annotation: disorder (Structural state)
Evidence: chromatography evidence used in manual assertion
Supporting statements:
The hydrodynamic radii (Stokes' radius, RS) of full-length P, PNTD, PCED, PCTD, PΔC120, and PΔ91–131 were determined by calibrating the SEC column with standard proteins.71 On a plot of the hydrodynamic radius as a function of molecular mass (Fig. 7b), PCED and PCTD appeared as native globular proteins, PΔ91–131, PΔC120, and full-length P were intermediate between native and unfolded proteins, indicating that these proteins are non-globular, and PNTD appeared as an unfolded protein.
Protein: DP01760 (UniProt P37173)
Region: residues 38	to 48
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Residues missing in electron density. Numbers in the manuscripts mentioning disorder in the N-terminal ligand binding domain always refer to mature protein lacking the signal peptide (residues 1-22).
Cross-references: PDB:1KTZ
Protein: DP01760 (UniProt P37173)
Region: residues 38	to 48
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The N- and C-terminal segments, residues 15−25 and 127−136, respectively, exhibit no detectable long-range NOE interactions and are structurally disordered (Figure 4B).
Cross-references: PDB:1PLO
Protein: DP01760 (UniProt P37173)
Region: residues 150	to 159
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The N- and C-terminal segments, residues 15−25 and 127−136, respectively, exhibit no detectable long-range NOE interactions and are structurally disordered (Figure 4B).
Cross-references: PDB:1PLO
Protein: DP01760 (UniProt P37173)
Region: residues 38	to 48
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The most striking difference lies with seven residues of the flexible N-terminal extension of TβRII that become ordered in the ternary complex, providing extensive receptor-receptor contact (Figure 2A and Figure S2).
Cross-references: PDB:2PJY
Protein: DP01760 (UniProt P37173)
Region: residues 38	to 48
Annotation: protein binding (Molecular function)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The bulk of receptor-receptor interaction arises from seven residues of the disordered N-terminal extension of TβRII that become ordered in the ternary complex and tether TβRI to its docking site at the composite interface.
Protein: DP01760 (UniProt P37173)
Region: residues 38	to 48
Annotation: molecular adaptor activity (Molecular function)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The disordered N-terminal extension of TβRII becomes partially structured in the complex, tethering TβRI to its docking site at the composite interface.
Protein: DP01760 (UniProt P37173)
Region: residues 38	to 48
Annotation: disorder to order (Structural transition)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The most striking difference lies with seven residues of the flexible N-terminal extension of TβRII that become ordered in the ternary complex, providing extensive receptor-receptor contact (Figure 2A and Figure S2).
Protein: DP01762 (UniProt P03714)
Region: residues 1	to 24
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
It also displays two large unstructured regions at the N terminus (residues 1–24) and in a large loop near the middle of the protein (residues 46–62).
The structure of gpFII consists of a single domain, with two large disordered regions; one at the N terminus (residues 1–24), and one in the middle of the protein (residues 46–62), both of which are characterized by no medium or long range NOEs.
Cross-references: PDB:1K0H
Protein: DP01762 (UniProt P03714)
Region: residues 46	to 62
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
It also displays two large unstructured regions at the N terminus (residues 1–24) and in a large loop near the middle of the protein (residues 46–62).
The structure of gpFII consists of a single domain, with two large disordered regions; one at the N terminus (residues 1–24), and one in the middle of the protein (residues 46–62), both of which are characterized by no medium or long range NOEs.
Cross-references: PDB:1K0H
Protein: DP01762 (UniProt P03714)
Region: residues 1	to 24
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Unstructured regions of gpFII (Fig. S2A) and gpV (3) were delineated by measuring {1H}-15N heteronuclear NOEs.
The NMR solution structure of gpFII displays significant disorder. (A) Values of the heteronuclear {1H}15N NOE as a function of position in gpFII. Residues that fall below the red horizontal line representing a cutoff of +0.65 show increased dynamics on the fast, ns-ps time scale (1). These include residues 1–24, 46–62, and 109–117.
In this model, the large N-terminal unstructured region (residues 1–24) of gpFII protrudes from one side of the ring whereas much of the central β3–β4 unstructured region (residues 46–62) is positioned on the opposite side.
Cross-references: PDB:2KX4
Protein: DP01762 (UniProt P03714)
Region: residues 46	to 62
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Unstructured regions of gpFII (Fig. S2A) and gpV (3) were delineated by measuring {1H}-15N heteronuclear NOEs.
The NMR solution structure of gpFII displays significant disorder. (A) Values of the heteronuclear {1H}15N NOE as a function of position in gpFII. Residues that fall below the red horizontal line representing a cutoff of +0.65 show increased dynamics on the fast, ns-ps time scale (1). These include residues 1–24, 46–62, and 109–117.
In this model, the large N-terminal unstructured region (residues 1–24) of gpFII protrudes from one side of the ring whereas much of the central β3–β4 unstructured region (residues 46–62) is positioned on the opposite side.
Cross-references: PDB:2KX4
Protein: DP01762 (UniProt P03714)
Region: residues 53	to 61
Annotation: virion assembly (Biological process)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
One of the most strongly dominant-negative mutants, Δ53–61, was titrated into a reaction containing a constant level of WT protein (Fig. S4). The inhibitory effect of this mutant was found to be strongly dose-dependent, and the curve of inhibition versus protein concentration could be fit by an exponential function assuming a simple competition between WT and mutant gpFII molecules. Fitting of these curves indicated that, if one third of the gpFII molecules incorporated into a phage were mutant, assembly would be inhibited. Further in vitro activity assays and EM experiments clearly showed that the Δ53–61 mutant inhibited phage assembly through its ability to bind heads, but not tails (SI Methods and Tables S2 and S3).
Taken together, the behavior of the gpFII mutants supports our model of the oligomeric structure of gpFII within phage particles.
Protein: DP01762 (UniProt P03714)
Region: residues 53	to 61
Annotation: virion assembly (Biological process)
Evidence: transmission electron microscopy evidence used in manual assertion
Supporting statements:
One of the most strongly dominant-negative mutants, Δ53–61, was titrated into a reaction containing a constant level of WT protein (Fig. S4). The inhibitory effect of this mutant was found to be strongly dose-dependent, and the curve of inhibition versus protein concentration could be fit by an exponential function assuming a simple competition between WT and mutant gpFII molecules. Fitting of these curves indicated that, if one third of the gpFII molecules incorporated into a phage were mutant, assembly would be inhibited. Further in vitro activity assays and EM experiments clearly showed that the Δ53–61 mutant inhibited phage assembly through its ability to bind heads, but not tails (SI Methods and Tables S2 and S3).
Taken together, the behavior of the gpFII mutants supports our model of the oligomeric structure of gpFII within phage particles.