LiteFold/DisProt huggingface dataset:
Protein: DP01546 (UniProt P84051)
Region: residues 98	to 124
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Chemical shift indices obtained from the experimental Cα and Cβ chemical shifts confirm the presence of the histone fold core secondary structure elements with disordered tails for both histones, similar to the human dimer (Fig. 1a).
Cross-references: PDB:7PJ1
Protein: DP01546 (UniProt P84051)
Region: residues 1	to 11
Annotation: flexible N-terminal tail (Disorder function)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Chemical shift indices obtained from the experimental Cα and Cβ chemical shifts confirm the presence of the histone fold core secondary structure elements with disordered tails for both histones, similar to the human dimer (Fig. 1a).
Cross-references: PDB:2PYO
Protein: DP01547 (UniProt P29274)
Region: residues 293	to 412
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The far-UV CD spectrum of A2A-ctL (residues 293-412) is typical for a protein without a globular conformation or secondary structure. It has a small negative shoulder around 222 nm, a strong negative minimum around 200 nm, and no positive signals in the far-UV range.
Protein: DP01547 (UniProt P29274)
Region: residues 293	to 412
Annotation: disorder (Structural state)
Evidence: fluorescence evidence used in manual assertion
Supporting statements:
A2A-ct has a single tryptophan residue at position 346. According to fluorescence measurements, Trp-346 is exposed to an aqueous environment.
Protein: DP01547 (UniProt P29274)
Region: residues 293	to 412
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The two-dimensional 1H, 15N-HSQC spectrum of A2A-ctL shows poorly dispersed crosspeaks, with the proton chemical shifts ranging from 7.5 to 8.5 1H ppm, indicating that A2A-ct undergoes fast conformational dynamics in solution, which is typical for disordered proteins. According to chemical shifts, the A2A-ctL N-terminal transiently populates β-structures, whereas the remainder is partially α-helical.
Protein: DP01547 (UniProt P29274)
Region: residues 293	to 412
Annotation: disorder (Structural state)
Evidence: small-angle X-ray scattering evidence used in manual assertion
Supporting statements:
A2A-ctL alone was highly elongated. For a fully random polymer chain, the Rg and Dmax values can be estimated from amino acid chain length; for A2A-ctL (134 residues), these values would be 3.78 nm and 10.5 nm, respectively. These results suggest A2A-ctL behaves more or less like a random chain in solution, and is even slightly more elongated than expected for a random polymer, suggesting intramolecular repulsion.
Protein: DP01547 (UniProt P29274)
Region: residues 293	to 412
Annotation: disorder (Structural state)
Evidence: chromatography evidence used in manual assertion
Supporting statements:
We determined Stokes radii for the peaks corresponding to A2A-ctL, CaM, and A2A-ctL-CaM samples by gel filtration, using proteins with known Stokes radii as standards. In the presence of calcium, the calculated Stokes radii for A2A-ctL, CaM, and the A2A-ctL-CaM complex were 2.74, 2.49, and 3.48 nm, respectively. The relatively high Stokes radii values were understandable because of the disordered nature of A2A-ct.
Protein: DP01547 (UniProt P29274)
Region: residues 293	to 412
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
H(N), N, C(α), C(β) and C' assignments of the intrinsically disordered C-terminus of human adenosine A2A receptor
We have previously structurally characterized this C-terminal domain and its interaction with calmodulin. It was shown to be structurally disordered and flexible, and to bind calmodulin with high affinity in a calcium-dependent manner. Interaction with calmodulin takes place at the N-terminal end of the A2A C-terminal domain without major conformational changes in the latter. NMR was one of the biophysical methods used in the study. Here we present the H(N), N, C(α), C(β) and C' chemical shift assignments of the free form of the C-terminus residues 293-412, used in the NMR spectroscopic characterization of the domain.
For the assignment we employed a combination of common 3D HN-detected assignment spectra and 3D HN-detected experiments specially tailored for intrinsically disordered proteins (Mäntylahti et al. 2009; Hellman et al. 2014). Fast conformational averaging and chemical exchange and absence of structure-induced secondary chemical shifts result in a narrow HN chemical shift range as well as overlap of Cα and Cβ shifts, both commonly observed for IDPs.
Cross-references: BMRB:25445
Protein: DP01547 (UniProt P29274)
Region: residues 291	to 311
Annotation: calmodulin binding (Molecular function)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
To confirm the CaM-binding site on A2A-ctL and the importance of arginine residues for binding, we carried out binding competition experiments using a peptide corresponding to WT A2AR amino acids 291–311 and a peptide corresponding to A2AR amino acids 288–311, with six arginine to alanine mutations (Fig. 4, B and C, respectively). The WT peptide bound calmodulin as expected, because an extra protein band appeared on a native gel. This band was missing from the mutated peptide-CaM sample, demonstrating the importance of the mutated arginine residues for the interaction. When the amount of WT A2A peptide was increased from 3.7 pmol to 24.3 nmol, the peptide clearly displaced A2A-ctL from CaM. On the other hand, the mutated A2A peptide did not affect A2A-ctL-CaM binding, although some unbound CaM was present in samples containing the highest amount of the mutated peptide.
Protein: DP01547 (UniProt P29274)
Region: residues 293	to 320
Annotation: calmodulin binding (Molecular function)
Evidence: isothermal titration calorimetry evidence used in manual assertion
Supporting statements:
As previous studies have not provided information about stoichiometry and affinity of the A2AR-calmodulin interaction, we performed ITC experiments for CaM binding to A2A-ctL in the presence of calcium and EDTA (Fig. 5, A and B). The A2A-ctL-CaM interaction is a calcium-dependent exothermic binding reaction with a favorable enthalpy (−12.93 ± 0.13 kcal mol−1), but an unfavorable entropy (3.4 kcal mol−1). The dissociation constant (Kd) for the complex is 97.9 ± 9.3 nM and the stoichiometry 0.92 ± 0.04, indicating 1:1 complex formation (Table 1). In the presence of EDTA, no binding was observed. To confirm the CaM-binding site on A2A-ctL, we also titrated A2A-ctS with CaM in the presence of calcium (Fig. 5 C). In this case, much smaller exothermic peaks were observed. Consequently, the removal of amino acids 293–320 from A2A-ct clearly abolished high-affinity binding to CaM.
The C-terminal construct of the A2AR—the full-length domain (A2A-ctL) corresponds to region 293–412, while the null-binding domain (A2A-ctS) corresponds to region 321-412.
Protein: DP01547 (UniProt P29274)
Region: residues 293	to 320
Annotation: calmodulin binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Most significant chemical shift perturbations, in terms of disappearance of crosspeaks of the free A2A-ctL form upon addition of CaM, were observed in the proposed CaM binding region in the very N-terminal part of A2A-ctL (Fig. 3 B). This suggests that the CaM binding epitope of A2A-ctL comprises residues 293–321
The C-terminal construct of the A2AR—the full-length domain (A2A-ctL) corresponds to region 293–412, while the null-binding domain (A2A-ctS) corresponds to region 321-412.
Protein: DP01547 (UniProt P29274)
Region: residues 293	to 412
Annotation: lipid binding (Molecular function)
Evidence: surface plasmon resonance evidence used in manual assertion
Supporting statements:
Because A2A-ctL seemed to interact with negatively charged lipids, we studied the effect of CaM on the ability of A2A-ctL to bind lipid membranes. For this purpose, we immobilized a mixture of DMPC/DMPG on an L1 SPR sensor chip, and injected A2A-ctL over the lipid surface. A2A-ctL bound immediately to the lipids in a concentration-dependent manner, and no dissociation was observed (Fig. 6).
Protein: DP01547 (UniProt P29274)
Region: residues 293	to 320
Annotation: calmodulin binding (Molecular function)
Evidence: native protein gel electrophoresis evidence used in manual assertion
Supporting statements:
Although A2A-ctL bound to CaM, no extra bands were detected in either of the samples containing A2A-ctS and CaM. This suggests that the CaM-binding site on A2A-ct locates between amino acids 293 and 320. This region on A2A-ct contains many positively charged arginine residues that are thought to be involved in CaM binding (19). The observed calcium dependence of the binding was surprising, because in previous studies, calcium was not thought to be involved in the interaction (42).
Protein: DP01547 (UniProt P29274)
Region: residues 317	to 412
Annotation: self-assembly (Disorder function)
Evidence: gel-filtration evidence used in manual assertion
Supporting statements:
Homo-oligomerization of the human adenosine A 2A receptor is driven by the intrinsically disordered C-terminus
Herein, we focus on the human adenosine A 2A receptor (A 2A R), a model GPCR that forms oligomers both in vitro and in vivo. Combining experimental and computational approaches, we discover that the intrinsically disordered C-terminus of A 2A R drives receptor homo-oligomerization. The formation of A 2A R oligomers declines progressively with the shortening of the C-terminus. Multiple interaction types are responsible for A 2A R oligomerization, including disulfide linkages, hydrogen bonds, electrostatic interactions, and hydrophobic interactions. These interactions are enhanced by depletion interactions, giving rise to a tunable network of bonds that allow A 2A R oligomers to adopt multiple interfaces. This study uncovers the disordered C-terminus as a prominent driving factor for the oligomerization of a GPCR, offering important insight into the effect of C-terminus modification on receptor oligomerization of A 2A R and other GPCRs reconstituted in vitro for biophysical studies.
Using the SEC analysis described earlier (Figure 1), we evaluated the HMW oligomer and dimer levels of the A 2A R-DC variants relative to that of the A 2A R full-length-wild-type (FL-WT) control. Both the dimer and the total oligomer levels of A 2A R decreased progressively with the shortening of the C-terminus, with almost no oligomerization detected upon complete truncation of the C-terminus at site A316 (Figure 3B). This result shows that the C-terminus drives A 2A R oligomerization, with multiple potential interaction sites positioned along much of its length.
Protein: DP01547 (UniProt P29274)
Region: residues 354	to 359
Annotation: self-assembly (Disorder function)
Evidence: gel-filtration evidence used in manual assertion
Supporting statements:
Since segment 354–359 contains three consecutive charged residues ( 355 ERR 357 ; Figure 3A), which could be involved in electrostatic interactions, we hypothesized that this 355 ERR 357 cluster could strengthen inter-protomer A 2A R-A 2A R association. To test this hypothesis, residues 355 ERR 357 were substituted by 355 AAA 357 on A 2A R-FL-WT and A 2A R-N359DC to generate A 2A R-ERR:AAA variants (Figure 3C). We then compared the HMW oligomer and dimer levels of the resulting variants with controls (same A 2A R variants but without the ERR:AAA mutations). We found that the ERR:AAA mutations had varied effects on the dimer level: decreasing for A 2A R-FL-WT (ctrl: 0.49; ERR:AAA:0.29) but increasing for A 2A R-N359DC (ctrl: 0.33; ERR:AAA: 0.48) (Figure 3C). In contrast, the ERR:AAA mutations reduced the HMW oligomer level of both A 2A R-FL-WT (ctrl: 0.88; ERR:AAA: 0.66) and A 2A R-N359DC (ctrl: 0.68; ERR:AAA: 0.38) (Figure 3C). Consistently, the ERR:AAA mutation lowered the total oligomer level of both A 2A R-FL-WT (ctrl: 1.37; ERR:AAA: 0.94) and A 2A R-N359DC (ctrl:1.01; ERR:AAA: 0.85) (Figure 3C). These results suggest that the charged residues 355 ERR 357 participate in A 2A R oligomerization, with a greater effect in the context of a longer C-terminus and for forming higher-order oligomers. The question then arises as to what types of interactions are formed along the C-terminus that help stabilize A 2A R oligomerization.
Upon closer examination, we recognize that at the very high ionic strength of 0.95 M the increase in the dimer and total oligomer levels was robust for A 2A R-FL-WT, but less pronounced for A 2A R-FL-ERR:AAA (Figure 5). Furthermore, this high ionic strength even had an opposite effect on A 2A R-N359DC, with both its dimer and total oligomer levels abolished (Figure 5). These results indicate that the charged cluster 355 ERR 357 and the C-terminal segment after residue N359 promote the depletion interactions to drive A 2A R oligomerization. Taken together, we can conclude that A 2A R oligomerization is more robust when the C-terminus is fully present and the ionic strength higher, suggesting that depletion interactions via the C-terminus are strong driving factors of A 2A R oligomerization.
In particular, the 355 ERR 357 cluster of charged residues lies distal from the dimeric interface but still forms several salt bridges (Figure 7A, inset). This observation is supported by our experimental results showing that substituting this charged cluster with alanines reduces the total A 2A R oligomer levels (Figure 3C).
Protein: DP01547 (UniProt P29274)
Region: residues 293	to 412
Annotation: calmodulin binding (Molecular function)
Evidence: small-angle X-ray scattering evidence used in manual assertion
Supporting statements:
Both A2A-ctL and CaM were monomeric in solution (Fig. S4), and the molecular mass calculated for their mixture indicated quantitative complex formation with 1:1 stoichiometry, which is in line with the affinity and stability of the complex described previously.
Protein: DP01548 (UniProt Q8CJF7)
Region: residues 1018	to 2243
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Figure 2
In vivo localization of metazoan ELYS domains
Protein: DP01549 (UniProt Q8WYP5)
Region: residues 1018	to 2266
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Figure 2 In vivo localization of metazoan ELYS domains
Protein: DP01550 (UniProt J7FTI7)
Region: residues 93	to 162
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
A 2D 1H–15N HSQC spectrum recorded at a pH of 6.8 and temperature of 298 K of an 15N-labeled glycine-rich (HvGR) protein sample reveals that the glycine-rich domain of HvGR-RBP1 is unstructured in solution, displaying only broad resonances in the central “random coil” region of the spectrum.
Protein: DP01550 (UniProt J7FTI7)
Region: residues 93	to 162
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Spectral changes taking place upon addition of unlabeled N-HvGR-RBP1 to 15N-HvGR suggest that the two protein domains can interact in vitro in trans, although the biological significance of these interactions is unclear.
Protein: DP01550 (UniProt J7FTI7)
Region: residues 93	to 162
Annotation: DNA binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Nucleic acid interaction assays indicated that the GR domain of HvGR-RBP1 can bind to single-stranded RNA (ssRNA) in the absence of the RRM domain.
Protein: DP01551 (UniProt Q99523)
Region: residues 97	to 108
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Figure 1 in original article and Supplementary material figure 4 text.
Protein: DP01552 (UniProt P33763)
Region: residues 38	to 52
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Also see supplements. In both apo-S100A5 and Ca2-S100A5, the relaxation
rate measurements show large mobility on a time scale
shorter than the reorientation time (R1 increases, R2
decreases, the NOE decreases) in the hinge loop and for the
last residues at the C-terminus, thus indicating that such
regions may be largely unstructured.
Protein: DP01553 (UniProt D6PZY5)
Region: residues 86	to 156
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The significant amount of peak doubling suggests that the non-glycine residues in the GR experience different chemical environments in relatively stable different conformations of the GR. The lack of 1H chemical shift dispersion for the GR resonances indicates a lack of structure. The peak doubling, broadened GR peaks and the perturbations of RRM resonances suggest that the GR may transiently interacts with the RRM.
Protein: DP01553 (UniProt D6PZY5)
Region: residues 86	to 156
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Together, these observations strongly suggest that the GR of the full-length protein can associate specifically to the RRM domain in an intermolecular fashion.
Protein: DP01554 (UniProt P23443)
Region: residues 422	to 525
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
In the 1H-15N HSQC spectrum of uniformly 15N-labeled AID (res. 422-525) the majority of backbone amide proton resonances exhibited poor chemical shift dispersion (∼7.8-8.6 ppm), indicating substantial regions of structural disorder. In addition, the majority of resonances were either broadened or missing, indicating interconversion between multiple conformations within the “intermediate” spectroscopic timescale.
Protein: DP01554 (UniProt P23443)
Region: residues 422	to 525
Annotation: self-inhibition (Disorder function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The best characterized function of the C-terminal AID is inhibition of upstream kinase-mediated HM (T412) and T-loop (T252) phosphorylation events, required for S6K1 activation. Trans-addition of purified recombinant ‘disordered’ AID does indeed effectively inhibit S6K1 phosphorylation by PDK1.
Protein: DP01554 (UniProt P23443)
Region: residues 422	to 525
Annotation: molecular recognition display site (Disorder function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Multi-site Ser-Thr phosphorylation of AID (S434, S441, T444, and S447 in S6K1) destabilizes its autoinhibitory interaction with the kinase domain of S6K1.
Protein: DP01554 (UniProt P23443)
Region: residues 422	to 525
Annotation: disorder (Structural state)
Evidence: sodium dodecyl sulfate polyacrylamide gel electrophoresis evidence used in manual assertion
Supporting statements:
AID displayed anomalously slower mobility in SDS-PAGE (apparent molecular mass of 15 kDa), that is 1.25-fold higher than the molecular mass of 11,988 Da calculated from its amino acid sequence.
Protein: DP01554 (UniProt P23443)
Region: residues 422	to 525
Annotation: disorder (Structural state)
Evidence: chromatography evidence used in manual assertion
Supporting statements:
AID displayed anomalously faster mobility in Superdex™ 75 size exclusion chromatography (apparent molecular mass of 15 kDa), 1.25-fold higher than the molecular mass of 11,988 Da calculated from its amino acid sequence.
Protein: DP01554 (UniProt P23443)
Region: residues 75	to 84
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Residues 75–84 at the N terminus and residues 372–399 at the C terminus are disordered.
Cross-references: PDB:3A60
PDB:3A61
Protein: DP01554 (UniProt P23443)
Region: residues 130	to 140
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
In addition, residues 130–140 of the putative helices αB and αC and residues 243–255 of the putative activation loop are also disordered (Fig. 6B).
Cross-references: PDB:3A60
PDB:3A61
Protein: DP01554 (UniProt P23443)
Region: residues 243	to 254
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
In addition, residues 130–140 of the putative helices αB and αC and residues 243–255 of the putative activation loop are also disordered (Fig. 6B).
Cross-references: PDB:3A60
PDB:3A61
Protein: DP01554 (UniProt P23443)
Region: residues 379	to 399
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Residues 75–84 at the N terminus and residues 372–399 at the C terminus are disordered.
Cross-references: PDB:3A60
PDB:3A61
Protein: DP01554 (UniProt P23443)
Region: residues 250	to 255
Annotation: phosphorylation display site (Disorder function)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
At least three phosphorylation events appear necessary for activation of p70S6K1 at Thr-252, Ser-394, and Thr-412.
Cross-references: PDB:3A62
Protein: DP01554 (UniProt P23443)
Region: residues 373	to 399
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Missing electron density for this region.
Cross-references: PDB:3A62
Protein: DP01554 (UniProt P23443)
Region: residues 250	to 255
Annotation: regulation of kinase activity (Biological process)
Evidence: in vitro protein kinase assay evidence used in manual assertion
Supporting statements:
The dephosphorylated protein was found to be catalytically inactive. Some activity could be restored upon phosphorylation of Thr-252 with PDK1 (Fig. 3A).
Protein: DP01555 (UniProt Q9EPU7)
Region: residues 4	to 91
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
CD spectra of AJ18–KRAB(Ab) showed that ity contains mostly random coil (56% ∼ 65%) with a small degree of helix (35% ∼ 39%) but little β sheet (0 ∼ 5%). These data suggest that the KRAB domain alone may not be highly structured.
Protein: DP01555 (UniProt Q9EPU7)
Region: residues 4	to 91
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The HSQC spectra of His-AJ18 KRAB(Ab) shows that the majority of backbone amide resonances exhibit poor dispersion, indicating substantial regions of disordered structure, consistent with CD results. NOESY of the sample exhibited minimal NOE cross-peaks providing further evidence of lack of a well defined 3D conformation. The 1D 1H spectra further confirm the lack of structure for the KRAB fragment.