LiteFold/DisProt huggingface dataset:
Protein: DP00018 (UniProt P46527)
Region: residues 55	to 95
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Titration of RhoA into 13C/15N-labeled p27 resulted in 2D CoN spectra exhibiting extensive resonance broadening for a region in the N-terminus encompassing residues 55–95 (Fig. 2). The finding that this region of p27 mediates interactions with RhoA is consistent with our observations that p27-C does not detectably associate with RhoA (Supplemental Fig. 2).
Using NMR spectroscopy and GDP exchange assays, we have shown that a region in the N-terminal region of p27 mediates binding to RhoA and interferes with nucleotide exchange.
Protein: DP00018 (UniProt P46527)
Region: residues 1	to 50
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Electron density is only observable for p27 residues 51–91, which corresponds to the region that binds Cdk2.
Cross-references: PDB:5UQ3
Protein: DP00018 (UniProt P46527)
Region: residues 97	to 197
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Electron density is only observable for p27 residues 51–91, which corresponds to the region that binds Cdk2.
Cross-references: PDB:5UQ3
Protein: DP00018 (UniProt P46527)
Region: residues 22	to 97
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The NMR properties of p27ID at 30 °C are characteristic of an unfolded protein. 1H chemical-shift ranges were obtained from DQF COSY (pH 4.4) or TOCSY−HSQC (pH 7.0) spectra using gradients for solvent suppression. The Hα and HN chemical shifts at pH 4.4 span 3.9−4.6 and 7.8−8.6 ppm, respectively, and at pH 7.0 span 4.05−4.59 and 7.77−8.58 ppm, respectively (data not shown). These chemical-shift ranges are typical of those observed in unfolded proteins (28). The amide 1H−15N NOE is negative (approximately −1 to −3.9) for each of 64 resolved mainchain amide cross-peaks at pH 4.4 (from an expected total of 70; data not shown), indicating that the p27ID main chain is highly flexible with motion on a time scale characteristic of unfolded proteins (29, 30). 
NMR study of the inhibitory domain of p27 (p27ID), spanning residues 22-97.
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:
The CD spectra of p27ID in kinase-assay buffer at 30 and 60 °C are also characteristic of an unfolded protein (Figure 5). The minimum at 204 nm is indicative of an unfolded conformation, and the weak negative shoulder at 222 nm suggests the absence of significant amounts of helix or β-strand. The CD spectrum of p27ID at 25 °C is independent of pH in the range 4 to 7.5 and concentration in the range 20 μM to 1 mM (data not shown). The spectroscopic data collectively indicate that the p27 Cdk-inhibition domain is intrinsically disordered in isolation under conditions where the domain is functional as a kinase inhibitor, in contrast to the ordered structure adopted upon binding cyclin A-Cdk2 (Figure 1A). 
p27ID inhibits histone H1 phosphorylation by cyclin A-Cdk2 (25 mM HEPES, 50 mM NaCl, and 5 mM MgCl2, pH 7.5, 30 °C). Reactions contained 50 nM cyclin A-Cdk2 and 0 or 50 nM p27ID. The cyclin A-Cdk2 used here is over 90% active, as calibrated with staurosporine (16).
CD far-UV study of the inhibitory domain of p27 (p27ID), spanning residues 22-97.
Protein: DP00018 (UniProt P46527)
Region: residues 22	to 97
Annotation: disorder to pre-molten globule (Structural transition)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The CD spectra of p27ID in kinase-assay buffer at 30 and 60 °C are also characteristic of an unfolded protein (Figure 5). The minimum at 204 nm is indicative of an unfolded conformation, and the weak negative shoulder at 222 nm suggests the absence of significant amounts of helix or β-strand. The CD spectrum of p27ID at 25 °C is independent of pH in the range 4 to 7.5 and concentration in the range 20 μM to 1 mM (data not shown). The spectroscopic data collectively indicate that the p27 Cdk-inhibition domain is intrinsically disordered in isolation Local Helix Formation in the p27 Cdk-Inhibition Domain. CD spectroscopy indicates that p27ID becomes partially helical at lower temperature (Figure 5). The lack of a folded baseline and differing estimates of [θ]222 for a fully formed helix hinder quantitative determinations of helical content in largely unfolded proteins such as p27ID. Using the method of Chen et al. (19) and CDPro (20), the helix content of p27ID at 5 °C is estimated to be 10 and 16%, respectively. Both estimates are less than the total α and 310 helix content of 33% observed in the crystal structure of the domain bound to cyclin A-Cdk2 (9). The temperature dependence of the CD signal at 222 nm indicates that the helix is only marginally stable (Figure 6). Thus, although intrinsically disordered at physiological temperature, p27ID exhibits an inherent propensity to adopt helical structure.
CD far-UV study of the inhibitory domain of p27 (p27ID), spanning residues 22-97.
Protein: DP00018 (UniProt P46527)
Region: residues 22	to 97
Annotation: molecular function inhibitor activity (Molecular function)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
p27ID binds cyclin A-Cdk2 with a Kd of 8 ± 2 nM at 30 °C (Figure 9). The L41P mutant, which contains approximately half of the helix in unbound p27ID, binds with essentially the same affinity (Kd = 9 ± 1 nM at 30 °C) as p27ID. The K47P and A55P mutants, in which helix formation is essentially abolished, bind with slightly lower affinities (16 ± 3 and 13 ± 2 nM, respectively, at 30 °C). At 15 °C, where the helix is more populated, the Kd values for p27ID and L41P are 8 ± 1 and 10 ± 2 nM, respectively (data not shown). Thus, some correlation exists between loss of α-helix in the unbound inhibitor and an increase in Kd. However, the differences are very small in free energy terms (ΔΔG = 0.1 to 0.4 ± 0.4 kcal mol-1) as compared to the free energy of binding (ΔG = −11.4 ± 0.3 kcal mol-1 for p27ID).
Helix Stabilization with Alanine Substitutions. The importance of preformed local helix formation can be addressed by stabilizing helices with multiple alanine substitutions (37).2 Three sites in the α-helix (Gln 40, Asp 44, and Lys 47) were substituted simultaneously with Ala. These residues do not make quaternary contacts with cyclin A-Cdk2 in the crystal structure (9), and AGADIR (38, 39) predicts a 50−100% increase in helix content for residues 38−52 of the α-helix upon their simultaneous substitution with Ala. CD spectroscopy indicates that helix content of the E40A/D44A/K47A mutant is increased as envisaged (Figures 6 and 7), with an estimated helix content at 5 °C of 17 or 22% from [θ]222 or CDPro, respectively. The expected value would be 29% if the α-helix (but not the 310 helix) was fully formed in the mutant (Figure 1B) (9). The Kd of the E40A/D44A/K47A mutant for cyclin A-Cdk2 is increased slightly to 12 ± 3 nM (ΔΔG = 0.4 ± 0.2 kcal mol-1 relative to p27ID; Figure 9).
We conclude that the unbound p27 Cdk-inhibition domain is largely unfolded with a detectable conformational bias toward the α-helix formed upon binding cyclin A-Cdk2. This conformational bias is not beneficial in thermodynamic or kinetic terms for productive folding and binding. Indeed, enhancement of the propensity to form the α-helix impedes kinase inhibition in kinetic terms.
Protein: DP00018 (UniProt P46527)
Region: residues 25	to 93
Annotation: disorder to order (Structural transition)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
The crystal structure of the human p27Kip1 kinase inhibitory domain bound to the phosphorylated cyclin A-cyclin-dependent kinase 2 (Cdk2) complex has been determined at 2.3 angstrom. p27Kip1 binds the complex as an extended structure interacting with both cyclin A and Cdk2. On cyclin A, it binds in a groove formed by conserved cyclin box residues. On Cdk2, it binds and rearranges the amino-terminal lobe and also inserts into the catalytic cleft, mimicking ATP.
Cross-references: PDB:1JSU
Protein: DP00018 (UniProt P46527)
Region: residues 25	to 93
Annotation: protein binding (Molecular function)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
The crystal structure of the human p27Kip1 kinase inhibitory domain bound to the phosphorylated cyclin A-cyclin-dependent kinase 2 (Cdk2) complex has been determined at 2.3 angstrom. p27Kip1 binds the complex as an extended structure interacting with both cyclin A and Cdk2. On cyclin A, it binds in a groove formed by conserved cyclin box residues. On Cdk2, it binds and rearranges the amino-terminal lobe and also inserts into the catalytic cleft, mimicking ATP.
Complexes of cyclins with cyclin-dependent kinases (CDKs) play a central role in the control of the eukaryotic cell cycle. The discovery of proteins that bind to and inhibit the catalytic activity of cyclin-CDK complexes has identified kinase inhibition as an intrinsic component of cell-cycle control (reviewed in refs 1-3). These inhibitors (CKIs) induce cell-cycle arrest in response to anti-proliferative signals, including contact inhibition and serum deprivation, TGFβ (ref. 5), myogenic, myeloid and neuronal differentiation, and DNA-damage checkpoints. The inhibitors, which are present in proliferating cells as well, may also help to coordinate cell-cycle progression by their redistribution between different cyclin-CDK complexes.
The inhibitory domain of p27 binds the cyclin A-Cdk2 complex as an extended structure interacting with both subunits. Key hydrophobic and hydrogen-bond interactions between a 10-amino-acid region of p27, which contains the conserved LFG sequence motif, and a  shallow groove of cyclin A, which consists of conserved cyclin-box residues, are likely to serve as an anchor point in initial binding, facilitating subsequent interactions with Cdk2. p27 bind-ing inhibits the CDK catalytic activity because its interactions with the N-terminal β-sheet of Cdk2 induce conformational changes that change the shape of the catalytic cleft and because p27 inserts into and fills up the catalytic cleft, eliminating any potential for ATP binding.
Cross-references: PDB:1JSU
Protein: DP00018 (UniProt P46527)
Region: residues 25	to 93
Annotation: molecular adaptor activity (Molecular function)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
The crystal structure of the human p27Kip1 kinase inhibitory domain bound to the phosphorylated cyclin A-cyclin-dependent kinase 2 (Cdk2) complex has been determined at 2.3 angstrom. p27Kip1 binds the complex as an extended structure interacting with both cyclin A and Cdk2. On cyclin A, it binds in a groove formed by conserved cyclin box residues. On Cdk2, it binds and rearranges the amino-terminal lobe and also inserts into the catalytic cleft, mimicking ATP.
Complexes of cyclins with cyclin-dependent kinases (CDKs) play a central role in the control of the eukaryotic cell cycle. The discovery of proteins that bind to and inhibit the catalytic activity of cyclin-CDK complexes has identified kinase inhibition as an intrinsic component of cell-cycle control (reviewed in refs 1-3). These inhibitors (CKIs) induce cell-cycle arrest in response to anti-proliferative signals, including contact inhibition and serum deprivation, TGFβ (ref. 5), myogenic, myeloid and neuronal differentiation, and DNA-damage checkpoints. The inhibitors, which are present in proliferating cells as well, may also help to coordinate cell-cycle progression by their redistribution between different cyclin-CDK complexes.
The inhibitory domain of p27 binds the cyclin A-Cdk2 complex as an extended structure interacting with both subunits. Key hydrophobic and hydrogen-bond interactions between a 10-amino-acid region of p27, which contains the conserved LFG sequence motif, and a  shallow groove of cyclin A, which consists of conserved cyclin-box residues, are likely to serve as an anchor point in initial binding, facilitating subsequent interactions with Cdk2. p27 bind-ing inhibits the CDK catalytic activity because its interactions with the N-terminal β-sheet of Cdk2 induce conformational changes that change the shape of the catalytic cleft and because p27 inserts into and fills up the catalytic cleft, eliminating any potential for ATP binding.
Cross-references: PDB:1JSU
Protein: DP00018 (UniProt P46527)
Region: residues 25	to 93
Annotation: cyclin-dependent protein kinase activating kinase regulator activity (Molecular function)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
The crystal structure of the human p27Kip1 kinase inhibitory domain bound to the phosphorylated cyclin A-cyclin-dependent kinase 2 (Cdk2) complex has been determined at 2.3 angstrom. p27Kip1 binds the complex as an extended structure interacting with both cyclin A and Cdk2. On cyclin A, it binds in a groove formed by conserved cyclin box residues. On Cdk2, it binds and rearranges the amino-terminal lobe and also inserts into the catalytic cleft, mimicking ATP.
Complexes of cyclins with cyclin-dependent kinases (CDKs) play a central role in the control of the eukaryotic cell cycle. The discovery of proteins that bind to and inhibit the catalytic activity of cyclin-CDK complexes has identified kinase inhibition as an intrinsic component of cell-cycle control (reviewed in refs 1-3). These inhibitors (CKIs) induce cell-cycle arrest in response to anti-proliferative signals, including contact inhibition and serum deprivation, TGFβ (ref. 5), myogenic, myeloid and neuronal differentiation, and DNA-damage checkpoints. The inhibitors, which are present in proliferating cells as well, may also help to coordinate cell-cycle progression by their redistribution between different cyclin-CDK complexes.
The inhibitory domain of p27 binds the cyclin A-Cdk2 complex as an extended structure interacting with both subunits. Key hydrophobic and hydrogen-bond interactions between a 10-amino-acid region of p27, which contains the conserved LFG sequence motif, and a  shallow groove of cyclin A, which consists of conserved cyclin-box residues, are likely to serve as an anchor point in initial binding, facilitating subsequent interactions with Cdk2. p27 bind-ing inhibits the CDK catalytic activity because its interactions with the N-terminal β-sheet of Cdk2 induce conformational changes that change the shape of the catalytic cleft and because p27 inserts into and fills up the catalytic cleft, eliminating any potential for ATP binding.
Cross-references: PDB:1JSU
ELM:DOC_CYCLIN_RxL_1
Protein: DP00018 (UniProt P46527)
Region: residues 181	to 185
Annotation: protein 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 Skp2.
Cross-references: PDB:2AST
ELM:DEG_SCF_SKP2-CKS1_1
Protein: DP00018 (UniProt P46527)
Region: residues 181	to 190
Annotation: disorder to order (Structural transition)
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.
Cross-references: PDB:2AST
Protein: DP00018 (UniProt P46527)
Region: residues 181	to 190
Annotation: ubiquitin ligase activator activity (Molecular function)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
The ubiquitin-mediated proteolysis of the Cdk2 inhibitor p27(Kip1) plays a central role in cell cycle progression, and enhanced degradation of p27(Kip1) is associated with many common cancers. Proteolysis of p27(Kip1) is triggered by Thr187 phosphorylation, which leads to the binding of the SCF(Skp2) (Skp1-Cul1-Rbx1-Skp2) ubiquitin ligase complex. Unlike other known SCF substrates, p27(Kip1) ubiquitination also requires the accessory protein Cks1.
The phosphorylated Thr187 side chain of p27Kip1 is recognized by a Cks1 phosphate binding site, whereas the side chain of an invariant Glu185 inserts into the interface between Skp2 and Cks1, interacting with both. The structure and biochemical data support the proposed model that Cdk2-cyclin A contributes to the recruitment of p27Kip1 to the SCFSkp2-Cks1 complex.
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.
Cross-references: ELM:DEG_SCF_SKP2-CKS1_1
Protein: DP00018 (UniProt P46527)
Region: residues 185	to 189
Annotation: phosphorylation display site (Disorder function)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
The ubiquitin-mediated proteolysis of the Cdk2 inhibitor p27(Kip1) plays a central role in cell cycle progression, and enhanced degradation of p27(Kip1) is associated with many common cancers. Proteolysis of p27(Kip1) is triggered by Thr187 phosphorylation, which leads to the binding of the SCF(Skp2) (Skp1-Cul1-Rbx1-Skp2) ubiquitin ligase complex. Unlike other known SCF substrates, p27(Kip1) ubiquitination also requires the accessory protein Cks1.
The phosphorylated Thr187 side chain of p27Kip1 is recognized by a Cks1 phosphate binding site, whereas the side chain of an invariant Glu185 inserts into the interface between Skp2 and Cks1, interacting with both. The structure and biochemical data support the proposed model that Cdk2-cyclin A contributes to the recruitment of p27Kip1 to the SCFSkp2-Cks1 complex.
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.
Evidence associated to the phosphorylated Thr187 of p27.
Cross-references: ELM:MOD_CDK_SPxK_1