LiteFold/DisProt huggingface dataset:
Protein: DP00015 (UniProt P61926)
Region: residues 15	to 24
Annotation: molecular adaptor activity (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Although the C-terminal tail of PKIα interacts transiently with the kinase’s C-lobe, it remains essentially disordered facilitating the recruitment by the CRM1/RanGTP complex for nuclear export.
The formation of the PKA-C/ATPγN/PKIα (ternary) complex is also reflected by the differences of longitudinal and transverse relaxation rates (ΔR1 and ΔR2) for both the HAR and PSS motifs upon forming the complex, which define a local rigidification of the PKIα backbone upon binding PKA-C, with the C-terminal portion (residues 47–75) remaining essentially unstructured (Figure 4B,C).
Region corresponding to the PSS motif of PKIα.
Protein: DP00015 (UniProt P61926)
Region: residues 15	to 24
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Although the C-terminal tail of PKIα interacts transiently with the kinase’s C-lobe, it remains essentially disordered facilitating the recruitment by the CRM1/RanGTP complex for nuclear export.
The formation of the PKA-C/ATPγN/PKIα (ternary) complex is also reflected by the differences of longitudinal and transverse relaxation rates (ΔR1 and ΔR2) for both the HAR and PSS motifs upon forming the complex, which define a local rigidification of the PKIα backbone upon binding PKA-C, with the C-terminal portion (residues 47–75) remaining essentially unstructured (Figure 4B,C).
Region corresponding to the PSS motif of PKIα.
Cross-references: BMRB:50243
Protein: DP00015 (UniProt P61926)
Region: residues 1	to 14
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Although the C-terminal tail of PKIα interacts transiently with the kinase’s C-lobe, it remains essentially disordered facilitating the recruitment by the CRM1/RanGTP complex for nuclear export.
The formation of the PKA-C/ATPγN/PKIα (ternary) complex is also reflected by the differences of longitudinal and transverse relaxation rates (ΔR1 and ΔR2) for both the HAR and PSS motifs upon forming the complex, which define a local rigidification of the PKIα backbone upon binding PKA-C, with the C-terminal portion (residues 47–75) remaining essentially unstructured (Figure 4B,C).
Region corresponding to the HAR motif of PKIα.
Cross-references: BMRB:50243
Protein: DP00015 (UniProt P61926)
Region: residues 1	to 14
Annotation: protein binding (Molecular function)
Evidence: small-angle X-ray scattering evidence used in manual assertion
Supporting statements:
In the bound state, the HAR motif of PKIα is more ordered and adopts a more stable helical conformation; whereas the NES motif is considerably more dynamic and adopts a transient helical conformation (Figure 7E).
Region corresponding to the HAR motif of PKIα.
Protein: DP00015 (UniProt P61926)
Region: residues 1	to 14
Annotation: pre-molten globule to molten globule (Structural transition)
Evidence: small-angle X-ray scattering evidence used in manual assertion
Supporting statements:
In the bound state, the HAR motif of PKIα is more ordered and adopts a more stable helical conformation; whereas the NES motif is considerably more dynamic and adopts a transient helical conformation (Figure 7E).
Region corresponding to the HAR motif of PKIα.
Protein: DP00015 (UniProt P61926)
Region: residues 1	to 76
Annotation: protein binding (Molecular function)
Evidence: fluorescence resonance energy transfer evidence used in manual assertion
Supporting statements:
The PKA-C/PKIα complex relaxes through two structurally and kinetically distinct states to form the complex
To determine the mechanism of binding PKA-C and PKIα, we utilized stopped-flow rapid mixing fluorescence resonance energy transfer (FRET) and analyzed transient and total changes in donor fluorescence during the binding reaction.
From an initial analysis of the binding curve, we found that the total fluorescence of the Alexa-488 donor emission from the PKA-CDONOR/PKIACCEPTOR changed bi-exponentially through the course of the binding reactions (Figure 6A–D). The bi-exponential behavior suggests the presence of an initial fast binding step followed by a slow structural rearrangement (Table 1; Gianni et al., 2014).
Multi-pathway mechanism of PKIα binding to PKA-C revealed by stopped-flow FRET experiments.
The pre-exponential amplitudes for the fast phases increased hyperbolically, while the slow phase decreased hyperbolically (Figure 6D–H). Taken together, the behavior of the rate constants and the pre-exponential amplitudes for the fast and slow phases are consistent with a multi-pathway mechanism in which there is a fast binding phase of PKIα ensembles competent for binding and a subsequent structural rearrangement of these conformers upon binding (Dogan et al., 2014; Gianni et al., 2014).
Protein: DP00015 (UniProt P61926)
Region: residues 1	to 76
Annotation: molecular adaptor activity (Molecular function)
Evidence: fluorescence resonance energy transfer evidence used in manual assertion
Supporting statements:
The PKA-C/PKIα complex relaxes through two structurally and kinetically distinct states to form the complex
To determine the mechanism of binding PKA-C and PKIα, we utilized stopped-flow rapid mixing fluorescence resonance energy transfer (FRET) and analyzed transient and total changes in donor fluorescence during the binding reaction.
From an initial analysis of the binding curve, we found that the total fluorescence of the Alexa-488 donor emission from the PKA-CDONOR/PKIACCEPTOR changed bi-exponentially through the course of the binding reactions (Figure 6A–D). The bi-exponential behavior suggests the presence of an initial fast binding step followed by a slow structural rearrangement (Table 1; Gianni et al., 2014).
Multi-pathway mechanism of PKIα binding to PKA-C revealed by stopped-flow FRET experiments.
The pre-exponential amplitudes for the fast phases increased hyperbolically, while the slow phase decreased hyperbolically (Figure 6D–H). Taken together, the behavior of the rate constants and the pre-exponential amplitudes for the fast and slow phases are consistent with a multi-pathway mechanism in which there is a fast binding phase of PKIα ensembles competent for binding and a subsequent structural rearrangement of these conformers upon binding (Dogan et al., 2014; Gianni et al., 2014).
Protein: DP00015 (UniProt P61926)
Region: residues 1	to 76
Annotation: molecular function inhibitor activity (Molecular function)
Evidence: sodium dodecyl sulfate polyacrylamide gel electrophoresis evidence used in manual assertion
Supporting statements:
The heat-stable inhibitor of the CAMP-dependent protein kinase is a small protein which binds to the catalytic subunit of the kinase and inhibits its activity (1, 2)
PKI was further purified by ion exchange and gel exclusion chromatography. Again, the purification was monitored by analysis of PKI activity (Fig. 2) and SDS-polyacrylamide gel electrophoresis (Fig. 3). Most of the PKI activity which could be recovered was found to elute in the 100-300 mM fraction on DEAE-chromatography, and gel electrophoresis suggested the presence of only minor contaminants at this stage of the purification. The minor contaminants were readily removed by chromatography on Sephadex G-50 yielding an apparently homogeneous preparation. The results of a typical purification are summarized in Table I. Starting from 6 liters of bacterial culture, a final preparation of 14 mg of purified PKI was obtained with an apparent overall yield of 9%. While analysis of activity suggests that only a 12.8-fold purification was necessary, this is probably an underestimate due to the presence of nonspecific inhibitory activity present in the crude extracts. The recovery was good for most steps, except for the Sephadex G-50 columns. Somewhat low recovery was encountered at this step because of the need to utilize only the peak fractions to avoid contamination with other small proteins. Characteristics of Bacterially Expressed PKI-The bacterially expressed PKI exhibited inhibitory potency identical to that of the native skeletal muscle protein. Both equally titrate the activity of pure CAMP-dependent protein kinase and exhibit identical specific activities (Fig. 4a). Henderson analysis (23) yielded K, values of 0.098 nM for the bacterial protein compared to 0.085 nM for the native protein; these values are identical within experimental error (Fig. 46)
The bacterially expressed PKI exhibited inhibitory potency identical to that of the native skeletal muscle protein. Both equally titrate the activity of pure CAMP-dependent protein kinase and exhibit identical specific activities (Fig. 4a)
The inhibitory activity of bacterially expressed PKI is indistinguishable from that of the native skeletal muscle protein (Fig. 4).
Gel electrophoresis characterization of bacterially-expressed and native PKIs. Analysis of PKI activity by SDS gel electrophoresis.
Protein: DP00016 (UniProt P38936)
Region: residues 1	to 164
Annotation: disorder (Structural state)
Evidence: sodium dodecyl sulfate polyacrylamide gel electrophoresis evidence used in manual assertion
Supporting statements:
Further, no difference in the protease maps was observed in the absence or presence of urea (2.0 M and 4.0 M), as judged using SDS/PAGE and HPLC (data not shown), providing further evidence that p21-F lacks stable structured domains.
Protein: DP00016 (UniProt P38936)
Region: residues 1	to 164
Annotation: disorder (Structural state)
Evidence: cleavage assay evidence used in manual assertion
Supporting statements:
Detailed analysis of trypsin cleavage products using MALDI mass spectrometry (22) showed that p21-F is cleaved at all predicted trypsin sites (Fig.1B) and reveals that globular protease-resistant domains do not exist.
Protein: DP00016 (UniProt P38936)
Region: residues 1	to 164
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
CD spectropolarimetry was used to investigate the secondary structure of the p21 NH2 terminus. CD spectra for p21-F, -A, and -B are very similar (Fig.3A) and indicate that (i) despite the clear demonstration of Cdk inhibitory activity, p21 and its fragments possess very little regular secondary structure, and (ii) the lack of secondary structure within the NH2-terminal fragments is not due to a requirement that COOH-terminal domains be present to promote a stable fold.
The CD data thus indicate that p21 does not exhibit the characteristic cooperative unfolding behavior expected of a globular protein but rather provide further evidence that the protein is highly flexible and disordered in the free solution state.
Protein: DP00016 (UniProt P38936)
Region: residues 1	to 94
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The 1H-15N correlation spectrum of p21-A (Fig.4A) reveals resonances that are clustered with in a narrow chemical shift range (total 1H chemical shift dispersion of backbone amides 0.80 ppm) and indicates that the chemical environment of each amide group, as reported by 1H and 15N chemical shifts, is influenced predominantly by the chemical nature of its own amino acid side chain and not by long-range effects due to secondary or tertiary structure. The spectrum lacks the chemical shift dispersion expected for a folded globular protein.
Protein: DP00016 (UniProt P38936)
Region: residues 9	to 84
Annotation: disorder to order (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
In sharp contrast to the disordered free state, however, the p21 NH2 terminus adopts an ordered stable conformation when bound to Cdk2, as shown directly by NMR spectroscopy. We have, thus, identified a striking disorder-order transition for p21 upon binding to one of its biological targets, Cdk2. This structural transition has profound implications in light of the ability of p21 to bind and inhibit a diverse family of cyclin-Cdk complexes, including cyclin A-Cdk2, cyclin E-Cdk2, and cyclin D-Cdk4.
The conformational state of p21-B, which is 18 amino acids shorter than p21-A but has similar biochemical activity (Fig.2), is dramatically changed upon addition of a stoichiometric amount of unlabeled Cdk2, as revealed by 1H-15N correlated spectra (Fig.5). Upon binding to Cdk2 (Fig.SB), there is a large increase in resonance dispersion (total 1H chemical shift dispersion of backbone amides ≈ 1.25 ppm).
Representative 15N line widths at half height for Cdk2-bound p21-B range from 9Hz to 16Hz, consistent with a monodisperse 44-kDa protein-protein complex.
Protein: DP00016 (UniProt P38936)
Region: residues 9	to 84
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
In sharp contrast to the disordered free state, however, the p21 NH2 terminus adopts an ordered stable conformation when bound to Cdk2, as shown directly by NMR spectroscopy. We have, thus, identified a striking disorder-order transition for p21 upon binding to one of its biological targets, Cdk2. This structural transition has profound implications in light of the ability of p21 to bind and inhibit a diverse family of cyclin-Cdk complexes, including cyclin A-Cdk2, cyclin E-Cdk2, and cyclin D-Cdk4.
The conformational state of p21-B, which is 18 amino acids shorter than p21-A but has similar biochemical activity (Fig.2), is dramatically changed upon addition of a stoichiometric amount of unlabeled Cdk2, as revealed by 1H-15N correlated spectra (Fig.5). Upon binding to Cdk2 (Fig.SB), there is a large increase in resonance dispersion (total 1H chemical shift dispersion of backbone amides ≈ 1.25 ppm).
Representative 15N line widths at half height for Cdk2-bound p21-B range from 9Hz to 16Hz, consistent with a monodisperse 44-kDa protein-protein complex.
Protein: DP00016 (UniProt P38936)
Region: residues 9	to 84
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
CD spectropolarimetry was used to investigate the secondary structure of the p21 NH2 terminus. CD spectra for p21-F, -A, and -B are very similar (Fig.3A) and indicate that (i) despite the clear demonstration of Cdk inhibitory activity, p21 and its fragments possess very little regular secondary structure, and (ii) the lack of secondary structure within the NH2-terminal fragments is not due to a requirement that COOH-terminal domains be present to promote a stable fold.
The CD data thus indicate that p21 does not exhibit the characteristic cooperative unfolding behavior expected of a globular protein but rather provide further evidence that the protein is highly flexible and disordered in the free solution state.
Fragment corresponding to p21-B.
Protein: DP00016 (UniProt P38936)
Region: residues 1	to 94
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
CD spectropolarimetry was used to investigate the secondary structure of the p21 NH2 terminus. CD spectra for p21-F, -A, and -B are very similar (Fig.3A) and indicate that (i) despite the clear demonstration of Cdk inhibitory activity, p21 and its fragments possess very little regular secondary structure, and (ii) the lack of secondary structure within the NH2-terminal fragments is not due to a requirement that COOH-terminal domains be present to promote a stable fold.
The CD data thus indicate that p21 does not exhibit the characteristic cooperative unfolding behavior expected of a globular protein but rather provide further evidence that the protein is highly flexible and disordered in the free solution state.
Fragment corresponding to p21-F.
Protein: DP00016 (UniProt P38936)
Region: residues 1	to 94
Annotation: molecular function inhibitor activity (Molecular function)
Evidence: enzymatic activity assay evidence used in manual assertion
Supporting statements:
The inhibitory activity of p21-F, -A, and -B toward Cdks was tested in vitro using cyclin A-Cdk2 purified from HeLa cells by immunoprecipitation and using histone H1 as the kinase substrate. The concentration producing 50% inhibition for all three p21 proteins is between 32 pM and 320 pM (Fig.2A). Cdk2 inhibitory activity was tested at 32 nM under the solution conditions used for structural studies (vide infra), including (i) high ionic strength, pH 7.0 (Fig.2A, B Left, and C Left), (ii) high ionic strength with sodium thiocyanate, pH 7.0 (Fig.2A, B Middle, and C Middle), and (iii) low ionic strength, pH 5.0 (Fig.2A, B Right, and C Right). Cdk2 kinase activity is diminished under conditions ii and iii compared with condition i (Fig.2B); significantly, however, p21-F, -A, and -B are potent Cdk2 inhibitors under all solution conditions (Fig.2C).
Fragment corresponding to p21-A.
Protein: DP00016 (UniProt P38936)
Region: residues 1	to 94
Annotation: protein binding (Molecular function)
Evidence: enzymatic activity assay evidence used in manual assertion
Supporting statements:
The inhibitory activity of p21-F, -A, and -B toward Cdks was tested in vitro using cyclin A-Cdk2 purified from HeLa cells by immunoprecipitation and using histone H1 as the kinase substrate. The concentration producing 50% inhibition for all three p21 proteins is between 32 pM and 320 pM (Fig.2A). Cdk2 inhibitory activity was tested at 32 nM under the solution conditions used for structural studies (vide infra), including (i) high ionic strength, pH 7.0 (Fig.2A, B Left, and C Left), (ii) high ionic strength with sodium thiocyanate, pH 7.0 (Fig.2A, B Middle, and C Middle), and (iii) low ionic strength, pH 5.0 (Fig.2A, B Right, and C Right). Cdk2 kinase activity is diminished under conditions ii and iii compared with condition i (Fig.2B); significantly, however, p21-F, -A, and -B are potent Cdk2 inhibitors under all solution conditions (Fig.2C).
Fragment corresponding to p21-A.
Protein: DP00016 (UniProt P38936)
Region: residues 9	to 84
Annotation: protein binding (Molecular function)
Evidence: enzymatic activity assay evidence used in manual assertion
Supporting statements:
The inhibitory activity of p21-F, -A, and -B toward Cdks was tested in vitro using cyclin A-Cdk2 purified from HeLa cells by immunoprecipitation and using histone H1 as the kinase substrate. The concentration producing 50% inhibition for all three p21 proteins is between 32 pM and 320 pM (Fig.2A). Cdk2 inhibitory activity was tested at 32 nM under the solution conditions used for structural studies (vide infra), including (i) high ionic strength, pH 7.0 (Fig.2A, B Left, and C Left), (ii) high ionic strength with sodium thiocyanate, pH 7.0 (Fig.2A, B Middle, and C Middle), and (iii) low ionic strength, pH 5.0 (Fig.2A, B Right, and C Right). Cdk2 kinase activity is diminished under conditions ii and iii compared with condition i (Fig.2B); significantly, however, p21-F, -A, and -B are potent Cdk2 inhibitors under all solution conditions (Fig.2C).
Fragment corresponding to p21-B.
Protein: DP00016 (UniProt P38936)
Region: residues 9	to 84
Annotation: molecular function inhibitor activity (Molecular function)
Evidence: enzymatic activity assay evidence used in manual assertion
Supporting statements:
The inhibitory activity of p21-F, -A, and -B toward Cdks was tested in vitro using cyclin A-Cdk2 purified from HeLa cells by immunoprecipitation and using histone H1 as the kinase substrate. The concentration producing 50% inhibition for all three p21 proteins is between 32 pM and 320 pM (Fig.2A). Cdk2 inhibitory activity was tested at 32 nM under the solution conditions used for structural studies (vide infra), including (i) high ionic strength, pH 7.0 (Fig.2A, B Left, and C Left), (ii) high ionic strength with sodium thiocyanate, pH 7.0 (Fig.2A, B Middle, and C Middle), and (iii) low ionic strength, pH 5.0 (Fig.2A, B Right, and C Right). Cdk2 kinase activity is diminished under conditions ii and iii compared with condition i (Fig.2B); significantly, however, p21-F, -A, and -B are potent Cdk2 inhibitors under all solution conditions (Fig.2C).
Fragment corresponding to p21-B.
Protein: DP00016 (UniProt P38936)
Region: residues 1	to 164
Annotation: molecular function inhibitor activity (Molecular function)
Evidence: enzymatic activity assay evidence used in manual assertion
Supporting statements:
The inhibitory activity of p21-F, -A, and -B toward Cdks was tested in vitro using cyclin A-Cdk2 purified from HeLa cells by immunoprecipitation and using histone H1 as the kinase substrate. The concentration producing 50% inhibition for all three p21 proteins is between 32 pM and 320 pM (Fig.2A). Cdk2 inhibitory activity was tested at 32 nM under the solution conditions used for structural studies (vide infra), including (i) high ionic strength, pH 7.0 (Fig.2A, B Left, and C Left), (ii) high ionic strength with sodium thiocyanate, pH 7.0 (Fig.2A, B Middle, and C Middle), and (iii) low ionic strength, pH 5.0 (Fig.2A, B Right, and C Right). Cdk2 kinase activity is diminished under conditions ii and iii compared with condition i (Fig.2B); significantly, however, p21-F, -A, and -B are potent Cdk2 inhibitors under all solution conditions (Fig.2C).
Fragment corresponding to p21-F.
Protein: DP00016 (UniProt P38936)
Region: residues 1	to 164
Annotation: protein binding (Molecular function)
Evidence: enzymatic activity assay evidence used in manual assertion
Supporting statements:
The inhibitory activity of p21-F, -A, and -B toward Cdks was tested in vitro using cyclin A-Cdk2 purified from HeLa cells by immunoprecipitation and using histone H1 as the kinase substrate. The concentration producing 50% inhibition for all three p21 proteins is between 32 pM and 320 pM (Fig.2A). Cdk2 inhibitory activity was tested at 32 nM under the solution conditions used for structural studies (vide infra), including (i) high ionic strength, pH 7.0 (Fig.2A, B Left, and C Left), (ii) high ionic strength with sodium thiocyanate, pH 7.0 (Fig.2A, B Middle, and C Middle), and (iii) low ionic strength, pH 5.0 (Fig.2A, B Right, and C Right). Cdk2 kinase activity is diminished under conditions ii and iii compared with condition i (Fig.2B); significantly, however, p21-F, -A, and -B are potent Cdk2 inhibitors under all solution conditions (Fig.2C).
Fragment corresponding to p21-F.
Protein: DP00016 (UniProt P38936)
Region: residues 1	to 164
Annotation: positive regulation of protein phosphorylation (Biological process)
Evidence: luciferase reporter gene assay evidence used in manual assertion
Supporting statements:
Surprisingly, we have found that p21 stimulates CDK8 kinase activity. p21 activation of CDK8, leading to transcriptional stimulation, stands in striking contrast to its inhibition of CDK2, the cell-cycle regulator primarily responsible for the ability of p21 to stop cell-cycle progression.
Protein: DP00016 (UniProt P38936)
Region: residues 1	to 164
Annotation: negative regulation of protein phosphorylation (Biological process)
Evidence: qualitative western immunoblotting evidence used in manual assertion
Supporting statements:
Thus, inactivation of Cdks by sustained expression of p21 may be the biochemical mechanism underlying the observed long-term cell cycle arrest.
Here, we present results that strongly suggest that this subunit mediates PCNA binding to DNA polymerase δ and corresponds to the subunit whose interaction with PCNA is targeted by p21Cip1.