LiteFold/DisProt huggingface dataset:
Protein: DP00018 (UniProt P46527)
Region: residues 185	to 190
Annotation: ubiquitin protein ligase binding (Molecular function)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
We obtained crystals of the ternary Skp1-Skp2-Cks1 complex by using a truncated human Cks1 (residues 5–73) and the previously described truncated Skp1-Skp2 complex (Schulman et al., 2000) (Figures 1A and 1B). For the crystallization of the quaternary Skp1-Skp2-Cks1-p27Kip1 complex, we used a 24 residue p27Kip1 phosphopeptide (residues 175–197; Figure 1C) that we show binds to Skp1-Skp2-Cks1 with an affinity essentially identical to that of full-length phosphorylated p27Kip1 (Table 1). This peptide is from the p27Kip1 C-terminal domain (CTD) that is distinct from the N-terminal domain (residues 22–106; NTD) that inhibits the Cdk2-cyclin A/E complexes (Russo et al., 1996).
In the structure of the quaternary Skp1-Skp2-Cks1-p27Kip1 complex, a ten amino acid segment of the p27Kip1 peptide (residues 181–190) adopts a partially extended conformation and spans the Skp2 LRR domain and Cks1 (Figure 2). The remaining 14 residues of the p27Kip1 peptide do not have interpretable electron density, and we presume they are disordered (Figure S1 available in the Supplemental Data with this article online). The N-terminal portion of the p27Kip1 segment packs with Skp2, a central Glu185 side chain inserts in between Skp2 and Cks1, and the C-terminal portion containing the phosphorylated Thr187 (pThr187) binds to Cks1. p27Kip1 binding does not cause any conformational changes in either Cks1 or Skp2.
The ten amino acid p27Kip1 segment binds to Skp2-Cks1 in a bipartite manner. Residues 181–184 bind to Skp2, residue 185 binds to both Skp2 and Cks1, and residues 186–190 bind to Cks1 (Figures 3C and 3D). The vast majority of the intermolecular contacts are centered on the Glu185 and pThr187 side chains of p27Kip1.
Region of p27 binding to Csk1.
Cross-references: PDB:2AST
ELM:DEG_SCF_SKP2-CKS1_1
Protein: DP00018 (UniProt P46527)
Region: residues 1	to 198
Annotation: localization (Biological process)
Evidence: deletion mutation phenotypic evidence used in manual assertion
Supporting statements:
The initial observation that the C-terminus of p27 is sufficient to co-immunoprecipitate with RhoA was made with a construct that was slightly longer than our p27-C construct [16]; however, extension of our construct to residue 88 (p2788–198) did not restore the migratory phenotype. For completeness, we also tested the complementary N-terminal construct, p271–87, which also did not exhibit a migratory phenotype. In summary, promotion of RhoA-dependent cell migration by p27 requires both the previously identified C-terminal region as well as the N-terminal region of p27 identified here.
Protein: DP00018 (UniProt P46527)
Region: residues 22	to 105
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
To further understand structure-function relationships for p27, we studied p27-FL and p27-KID using solution NMR spectroscopy. Comparison of 2D 1H-15N HSQC NMR spectra for the two polypeptides (Supplementary Fig. 1a,c online) corroborates findings based on CD spectra. The subspectrum in Supplementary Figure 1b online, showing only resonances that appear at the same chemical shift values in Supplementary Figure 1a,c online, shows ∼80% of the resonances observed for p27-KID (Supplementary Fig. 1c online). This illustrates that the conformation of most residues in p27-KID is virtually equivalent in the truncated and full-length molecules. Moreover, spectra for both molecules show limited resonance dispersion (backbone amide protons resonate between 7.8 and 8.5 p.p.m.), a feature typical of intrinsically disordered, biologically active polypeptides
p27-KID spans residues 22-105 of the human p27.
Protein: DP00018 (UniProt P46527)
Region: residues 1	to 198
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
To further understand structure-function relationships for p27, we studied p27-FL and p27-KID using solution NMR spectroscopy. Comparison of 2D 1H-15N HSQC NMR spectra for the two polypeptides (Supplementary Fig. 1a,c online) corroborates findings based on CD spectra. The subspectrum in Supplementary Figure 1b online, showing only resonances that appear at the same chemical shift values in Supplementary Figure 1a,c online, shows ∼80% of the resonances observed for p27-KID (Supplementary Fig. 1c online). This illustrates that the conformation of most residues in p27-KID is virtually equivalent in the truncated and full-length molecules. Moreover, spectra for both molecules show limited resonance dispersion (backbone amide protons resonate between 7.8 and 8.5 p.p.m.), a feature typical of intrinsically disordered, biologically active polypeptides
Protein: DP00018 (UniProt P46527)
Region: residues 28	to 37
Annotation: protein binding (Molecular function)
Evidence: isothermal titration calorimetry evidence used in manual assertion
Supporting statements:
Domain 1, though highly dynamic in solution, is relatively compact (ten residues), and therefore folding to a single conformation on binding cyclin A is associated with a relatively small entropy penalty. In contrast, domain 2 spans 29 residues that fold on binding to Cdk2, and, correspondingly, this reaction is associated with a large entropy penalty. Notably, domains 1 and 2 bind their respective targets with similar affinity, clearly revealing the thermodynamic importance of each of these interactions.
Our data show that interactions mediated by the two conserved domains of p27-KID contribute considerably to the overall ΔG value.
p27-KID binds Cdk2–cyclin A through a sequential mechanism involving extensive folding of p27-KID.
Evidence corresponding to domain 1 (28-37).
Protein: DP00018 (UniProt P46527)
Region: residues 66	to 88
Annotation: protein binding (Molecular function)
Evidence: isothermal titration calorimetry evidence used in manual assertion
Supporting statements:
Domain 1, though highly dynamic in solution, is relatively compact (ten residues), and therefore folding to a single conformation on binding cyclin A is associated with a relatively small entropy penalty. In contrast, domain 2 spans 29 residues that fold on binding to Cdk2, and, correspondingly, this reaction is associated with a large entropy penalty. Notably, domains 1 and 2 bind their respective targets with similar affinity, clearly revealing the thermodynamic importance of each of these interactions.
Our data show that interactions mediated by the two conserved domains of p27-KID contribute considerably to the overall ΔG value.
p27-KID binds Cdk2–cyclin A through a sequential mechanism involving extensive folding of p27-KID.
Evidence corresponding to domain 2 (66-88).
Protein: DP00018 (UniProt P46527)
Region: residues 32	to 45
Annotation: localization (Biological process)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
A putative nuclear export sequence (NES) in p27 is identified. Mutation of this NES reduced nuclear export, impaired p27-CRM1 interactions, and increased p27 stability.
p27 Contains an Atypical NES
We identified a putative NES within the Cdk-binding domain of p27 between amino acids 32 and 45 (Figure ​(Figure6A)6A) based on the homology of leucine spacing to a cryptic NES identified in the equine infectious anemia virus (EIAV) Rev protein (Mancuso et al., 1998 blue right-pointing triangle). The spacing of the three leucines in this region of p27 is highly conserved between species.
To obtain additional evidence that amino acids 32 through 45 in p27 comprise a functional NES, we tested its ability to mediate nuclear export of an unrelated protein. For this, peptides containing the putative p27 NES (CRNLFGPVDHEELTRDLE) were coupled to FITC-labeled BSA and were microinjected into nuclei of adherent HeLa cells. As is shown in Figure ​Figure6B,6B, a significant fraction of p27NES-FITC-BSA translocated into the cytoplasm within 45 min. In contrast, the nuclear localization of FITC-BSA remained unchanged. Although p27-NES mediated export of p27NES-FITC-BSA is not very efficient, possibly due to competing events such as the observed accumulation in nuclear speckles, or due to rate-limiting binding partners, these findings support the interpretation that amino acids 32 through 45 in p27 function as an NES.
p27NES Shows Delayed Cytoplasmic Accumulation after Proteasome Inhibition
Protein: DP00018 (UniProt P46527)
Region: residues 8	to 12
Annotation: nuclear export (Biological process)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
Mutation of S10 But Not of T187 Affects Nuclear Export of p27
Mutation of S10 to alanine strongly inhibited p27 export (Figure ​(Figure6,6, B and C), suggesting that phosphorylation at this site is essential for nuclear export of p27.
Protein: DP00018 (UniProt P46527)
Region: residues 8	to 12
Annotation: phosphorylation display site (Disorder function)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
Phosphorylation at serine 10, a major phosphorylation site of p27(Kip1), increases its protein stability
Substitution of Ser(10) with Ala (S10A) markedly reduced the extent of p27(Kip1) phosphorylation and prevented the shift in electrophoretic mobility. Phosphopeptide mapping and phosphoamino acid analysis revealed that phosphorylation at Ser(10) accounted for approximately 70% of the total phosphorylation of p27(Kip1), and the extent of phosphorylation at this site was approximately 25- and 75-fold greater than that at Ser(178) and Thr(187), respectively.
Furthermore, a mutant p27(Kip1) in which Ser(10) was replaced with glutamic acid in order to mimic the effect of Ser(10) phosphorylation exhibited a marked increase in stability both in vivo and in vitro compared with the wild-type or S10A mutant proteins. These results suggest that Ser(10) is the major site of phosphorylation of p27(Kip1) and that phosphorylation at this site, like that at Thr(187), contributes to regulation of p27(Kip1) stability.
Protein: DP00018 (UniProt P46527)
Region: residues 32	to 45
Annotation: phosphorylation display site (Disorder function)
Evidence: qualitative western immunoblotting evidence used in manual assertion
Supporting statements:
Delayed Nuclear Export Is Associated with Increased p27 Protein Stability
Protein: DP00018 (UniProt P46527)
Region: residues 22	to 105
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Before binding, p27 is at least partially disordered and folds upon binding its Cdk/cyclin targets.
Interestingly, the highly conserved, specificity determining segment of p27 is shown to be highly disordered.
In summary, these results provide strong evidence for the existence of IFSUs within localized regions of p27-KID; other segments of p27-KID are dynamic and disordered.
Protein: DP00018 (UniProt P46527)
Region: residues 38	to 90
Annotation: disorder to order (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Before binding, p27 is at least partially disordered and folds upon binding its Cdk/cyclin targets.
Interestingly, the highly conserved, specificity determining segment of p27 is shown to be highly disordered.
In summary, these results provide strong evidence for the existence of IFSUs within localized regions of p27-KID; other segments of p27-KID are dynamic and disordered. Importantly, while domain LH was known to be helical, by monitoring 1HN–1HN NOE correlations, we have detected additional structural features of p27-KID (IFSUs) that were not revealed through our previous NMR studies based on the analysis of Δδ13Cα and hetNOE values.
After domain 1 becomes tethered to the cyclin subunit of a Cdk/cyclin complex, the helical IFSU within domain LH positions domain 2 (containing domains 2.1, 2.2 and 2.3) for reorganization and folding on binding to Cdk2.
Protein: DP00018 (UniProt P46527)
Region: residues 22	to 97
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
In the presence of macromolecular crowding agents, neither the intrinsically disordered C-terminal activation domain of c-Fos nor the kinase-inhibition domain of p27Kip1 undergoes any significant conformational change that is detected by changes in either circular dichroism or fluorescence spectra.
While intrinsically disordered at physiological temperatures, marginally stable helix is present in p27ID at 5 °C (Figure 2A).8 Addition of TFE to p27ID induced a marked change in the CD spectrum indicative of a large increase in helix content (Figure 2A). However addition of several crowding agents did not induce any significant change in the CD spectrum of p27ID above 212 nm that reflects formation of helical or strand secondary structure (Figure 2B).
A decrease in the intensity of the CD signal of p27ID at 204 nm was observed in the presence of the 37.5 and 77 kDa dextrans, and the fluorescence emission intensity of p27ID at 350 nm was reduced in the presence of 77 kDa dextran and Ficoll 70 (as observed for FosAD).
Protein: DP00018 (UniProt P46527)
Region: residues 22	to 97
Annotation: disorder (Structural state)
Evidence: intrinsic fluorescence-based protein conformation evidence
Supporting statements:
In the presence of macromolecular crowding agents, neither the intrinsically disordered C-terminal activation domain of c-Fos nor the kinase-inhibition domain of p27Kip1 undergoes any significant conformational change that is detected by changes in either circular dichroism or fluorescence spectra.
Crowding agents did not affect the p27ID fluorescence emission maximum of 350 nm (Figure 2C) or did not affect significantly the fluorescence spectrum of ANS in the presence of p27ID (data not shown).
A decrease in the intensity of the CD signal of p27ID at 204 nm was observed in the presence of the 37.5 and 77 kDa dextrans, and the fluorescence emission intensity of p27ID at 350 nm was reduced in the presence of 77 kDa dextran and Ficoll 70 (as observed for FosAD).
Protein: DP00018 (UniProt P46527)
Region: residues 22	to 97
Annotation: protein binding (Molecular function)
Evidence: intrinsic fluorescence-based protein conformation evidence
Supporting statements:
Before binding, p27 is at least partially disordered and folds upon binding its Cdk/cyclin targets.
After domain 1 becomes tethered to the cyclin subunit of a Cdk/cyclin complex, the helical IFSU within domain LH positions domain 2 (containing domains 2.1, 2.2 and 2.3) for reorganization and folding on binding to Cdk2.
The need for structural rearrangement of domain 2.2 of p27-KID and the aforementioned segment of Cdk2 prior to binding Cdk2/cyclin A is likely the principal determinant of the slow kinetics of this interaction.
Protein: DP00018 (UniProt P46527)
Region: residues 86	to 90
Annotation: phosphorylation display site (Disorder function)
Evidence: qualitative western immunoblotting evidence used in manual assertion
Supporting statements:
A conserved tyrosine residue (Y88) in the Cdk-binding domain of p27 can be phosphorylated by the Src-family kinase Lyn and the oncogene product BCR-ABL.
p27 is phosphorylated on Y88 in vivo. HA-tagged p27 was coexpressed in 293T cells with Lyn (Y508F) or p210 BCR-ABL kinase. Anti-HA immunoprecipitates were incubated with (+) or without calf intestinal alkaline phosphatase (CIAP) and analyzed by western blotting using purified polyclonal anti-phospho-Y88 p27 antibodies. The same blot was subsequently probed with an HRP-conjugated anti-p27 antibody.
pY88 was annotated with 2-residue flanking regions in both directions.
Protein: DP00018 (UniProt P46527)
Region: residues 86	to 90
Annotation: phosphorylation display site (Disorder function)
Evidence: radioisotope assay evidence used in manual assertion
Supporting statements:
A conserved tyrosine residue (Y88) in the Cdk-binding domain of p27 can be phosphorylated by the Src-family kinase Lyn and the oncogene product BCR-ABL.
(D) Lyn and Abl phosphorylate Y88 of p27 in vitro. Equal amounts of p27 or mutant p27 (tyrosine residues exchanged to phenylalanine; Y1F: Y in position 74 exchanged to F; Y2: Y in position 88; and Y3: Y in position 89) were incubated with recombinant constitutively active Lyn or Abl kinase at 30°C in the presence of γ-[32P]-ATP. Proteins were separated by SDS-PAGE and detected by Coomassie staining (upper panel). The amount of incorporated 32P was shown by autoradiography (lower panel).
pY88 was annotated with 2-residue flanking regions in both directions.
Protein: DP00018 (UniProt P46527)
Region: residues 91	to 96
Annotation: protein binding (Molecular function)
Evidence: qualitative western immunoblotting evidence used in manual assertion
Supporting statements:
A conserved tyrosine residue (Y88) in the Cdk-binding domain of p27 can be phosphorylated by the Src-family kinase Lyn and the oncogene product BCR-ABL.
Within the Cdk-inhibitory domain the p27 sequence contains three tyrosine residues near the proline-rich SH3-binding element (Figure 1A). To investigate if phosphorylation of tyrosines influenced binding of p27 to the kinase, we exchanged these tyrosine residues to phenylalanine.
Figure 1  p27Kip1 Binds the Tyrosine Kinase Lyn and Is Phosphorylated on Tyrosine. (A) All tyrosine residues (indicated as Y) of p27 are located in the Cdk-inhibitory domain (dark gray). A proline-rich SH3-binding element (residues 91–96) is represented as a black box. (B) Lyn binds to a p27 mutant protein lacking all three tyrosines.
Equal amounts of monomeric or cyclin A/Cdk2-bound p27 were incubated with Abl kinase. While monomeric p27 was a better substrate for Abl phosphorylation, tyrosine phosphorylation of cyclin A/Cdk2-bound p27 was clearly detected (Figure 1G).
Protein: DP00018 (UniProt P46527)
Region: residues 58	to 105
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
These RD results, we believe for the first time, define the kinetics for transitions between discrete conformational states for a region of an IDP (p27-D2).
The RD data recorded with the lowest ωeff values (with maximal contributions of conformational exchange) revealed elevated R2,eff values for residues within several regions of the p27-D2 sequence, especially those containing aromatic residues (Figure 1C, gray points). In contrast, the apparent R2,0 (R2,0app) values (Figure 1C, black points) for p27-D2, which were determined from the RD fit procedure, are relatively continuous with few extrema showing that only the relaxation effects of fast segmental motions are retained (as expected for a disordered polypeptide) and that the contribution of Rex is largely quenched.
Protein: DP00018 (UniProt P46527)
Region: residues 58	to 105
Annotation: disorder (Structural state)
Evidence: small-angle X-ray scattering evidence used in manual assertion
Supporting statements:
Analysis using small-angle X-ray scattering (SAXS) (58, 59) showed that the radius of gyration (Rg) for p27-D2 increased from 21 ± 2 to 27 ± 2 Å in the presence of SJ403 ([p27-D2]:[SJ403] ratio of 1:5; Figure 4C), consistent with our hypothesis.
For a 48-residue-long polypeptide chain, Rg of 21 Å is significantly higher than the Rg of a globular protein of this size, 21 Å is characteristic of a typical IDP.
Protein: DP00018 (UniProt P46527)
Region: residues 1	to 60
Annotation: protein binding (Molecular function)
Evidence: yeast 2-hybrid evidence used in manual assertion
Supporting statements:
The pVHL β-domain is able to bind any member of the CDKN1 family, as indicated by yeast cell growth (Fig. 4B, Supplementary Figures S5 and S7).
Yeast plasmids expressing either their N-terminal tail containing the CDI domain (p27-NT residues: 1–60; p21-NT: 1–49; p57-NT: 1–61) or the corresponding C-terminal moiety lacking the N-terminus (p27-ΔN residues: 61–198; p21-ΔN: 50–164; p57-ΔN: 62–316) were generated to test the effects of CDKN1 binding to pVHL30. As shown in Fig. 4D, loss of the p27 N-terminus clearly disrupts its ability to associate with pVHL30, as yeast cells expressing the C-terminus of p27 were all unable to grow in selective medium.
Cross-references: DisProt:DP00287r060
Protein: DP00018 (UniProt P46527)
Region: residues 1	to 198
Annotation: protein binding (Molecular function)
Evidence: co-immunoprecipitation evidence used in manual assertion
Supporting statements:
Our data (Fig. 2B) indicates that all three CDKN1 proteins were able to interact with pVHL30, as demonstrated by their presence in the immunoprecipitate revealed with the anti-Flag antibody (bottom panels). Taken together, the experiments show that these proteins can form at least binary complexes in human, and notably kidney cells.
Cross-references: DisProt:DP00287r063
Protein: DP00020 (UniProt P11938)
Region: residues 1	to 123
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The solution structure of Rap1 BRCT domain contains three β-strands and three α-helices. Three helices, α1 (residue 31–39), α2 (residue 75–81) and α3 (residue 87–90), face the β-sheet that is formed by strand 1 (residue 16–18), strand 2 (residue 47–48) and strand 3 (residue 59–60).
The proportion of the secondary structure elements in Rap1 BRCT domain is relatively low and there are several long flexible loops among secondary structure elements. Therefore, the global conformation of Rap1 BRCT domain is rather flexible. No resonance signal maintained in 2-D 1H–2H exchange experiment also indicated a loose structure of Rap1 BRCT domain.
Cross-references: PDB:2L42
BMRB:17212
Protein: DP00020 (UniProt P11938)
Region: residues 482	to 512
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The resulting electron density map was interpretable for the DNA and the protein, with the exception of residues at the termini (residues 353–359 and 595–598), three internal regions (residues 482–512, 565–571, and 579–585), and one of the overhanging bases of the binding site.
Cross-references: PDB:1IGN
Protein: DP00020 (UniProt P11938)
Region: residues 627	to 675
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The PDB shows this region lacks electron density, indicating it is disordered.
Cross-references: PDB:4BJT
Protein: DP00020 (UniProt P11938)
Region: residues 565	to 598
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The resulting electron density map was interpretable for the DNA and the protein, with the exception of residues at the termini (residues 353–359 and 595–598), three internal regions (residues 482–512, 565–571, and 579–585), and one of the overhanging bases of the binding site.
Although the regions 572-578 and 586-594 have electron density in the PDB, they are not large enough to be ordered, so are considered as unstructured
Cross-references: PDB:1IGN
Protein: DP00020 (UniProt P11938)
Region: residues 627	to 674
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The PDB shows this region lacks electron density, indicating it is disordered.
Cross-references: PDB:4BJ6
Protein: DP00021 (UniProt P13551)
Region: residues 40	to 64
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The PDB shows this region lacks electron density, indicating it is disordered.
Cross-references: PDB:2BM1