LiteFold/DisProt huggingface dataset:
Protein: DP02945 (UniProt Q96RD7)
Region: residues 374	to 426
Annotation: flexible C-terminal tail (Disorder function)
Evidence: cryogenic electron microscopy evidence used in manual assertion
Supporting statements:
The PANX1 channel forms a heptamer that contains (from top to bottom) an extracellular domain (ECD), a transmembrane domain (TMD) and an intracellular domain (ICD), with the unstructured CTT blocking the ICD entrance to the ion pathway along the symmetry axis (referred to as the main pore) (Fig. 1a, b).
Although we were unable to model the CTT owing to its intrinsically disordered nature, we have defined its key role in caspase-dependent channel gating (see ‘Ion pathways and channel gating’).
Instead, we observed that the intracellular entry was blocked by the unstructured CTT, and this blockage was released upon removal of the CTT by caspase 7, opening the main pore (Fig. 1c).
Under normal physiological conditions, the intracellular entrance of the main pore is blocked by the unstructured CTT, so anions—but not ATP—can move through the side tunnels, which explains why PANX1 currents can be evoked with a voltage clamp, during which there is no ATP release
Protein: DP02946 (UniProt Q2VL32)
Region: residues 278	to 314
Annotation: disorder (Structural state)
Evidence: cleavage assay evidence used in manual assertion
Supporting statements:
The primary structure of AIP56 suggests that this toxin comprises two functional domains and could be an A-B toxin with its two moieties linked by a single disulphide bond (Figure S1) [23]. Therefore, in order to define domain boundaries within the toxin, limited proteolysis experiments were performed. SDS-PAGE analysis of AIP56 digested with chymotrypsin, trypsin or proteinase K revealed that the toxin is highly resistant to trypsin digestion, whereas chymotrypsin and proteinase K cleaved AIP56 into two major fragments with approximately 32 and 24 kDa (Figure 2A). These two fragments were only detected upon treatment with the reducing agent DTT, suggesting that they are linked by a disulphide bridge (Figure 2B). N-terminal Edman sequencing revealed that chymotrypsin cleavage occurred between Phe285 and Phe286, in the amino-acid stretch flanked by the two unique cysteine residues (Cys262 and Cys298) of AIP56 (Figure 2C). Altogether, these results indicate that AIP56 is composed of two domains linked by a disulphide bridge.
The region described in the publication, spanning residues Cys262 to Cys298, corresponds to region Cys278-Cys314 of the associated UniProt sequence as AIP56 was synthesized as a precursor protein with a cleavable N-terminal signal peptide that was removed during secretion, originating a 497-amino acid mature toxin.
Protein: DP02946 (UniProt Q2VL32)
Region: residues 278	to 314
Annotation: flexible linker (Disorder function)
Evidence: cleavage assay evidence used in manual assertion
Supporting statements:
The primary structure of AIP56 suggests that this toxin comprises two functional domains and could be an A-B toxin with its two moieties linked by a single disulphide bond (Figure S1) [23]. Therefore, in order to define domain boundaries within the toxin, limited proteolysis experiments were performed. SDS-PAGE analysis of AIP56 digested with chymotrypsin, trypsin or proteinase K revealed that the toxin is highly resistant to trypsin digestion, whereas chymotrypsin and proteinase K cleaved AIP56 into two major fragments with approximately 32 and 24 kDa (Figure 2A). These two fragments were only detected upon treatment with the reducing agent DTT, suggesting that they are linked by a disulphide bridge (Figure 2B). N-terminal Edman sequencing revealed that chymotrypsin cleavage occurred between Phe285 and Phe286, in the amino-acid stretch flanked by the two unique cysteine residues (Cys262 and Cys298) of AIP56 (Figure 2C). Altogether, these results indicate that AIP56 is composed of two domains linked by a disulphide bridge.
AIP56 toxicity requires integrity of the linker but the disulfide bridge is dispensable for intoxication.
The region described in the publication, spanning residues Cys262 to Cys298, corresponds to region Cys278-Cys314 of the associated UniProt sequence as AIP56 was synthesized as a precursor protein with a cleavable N-terminal signal peptide that was removed during secretion, originating a 497-amino acid mature toxin.
Protein: DP02946 (UniProt Q2VL32)
Region: residues 278	to 314
Annotation: localization (Biological process)
Evidence: sodium dodecyl sulfate polyacrylamide gel electrophoresis evidence used in manual assertion
Supporting statements:
Translocation of AIP56 requires integrity of the Cys262-Cys298 linker but the disulphide bridge is not an absolute requirement for toxicity
These results suggest that the integrity of the linker region between the two cysteine residues is needed for toxin internalization, in contrast to what is known for the diphtheria, tetanus and botulinum toxins, where nicking of the inter-cysteine loop is required for toxicity
In contrast, we have found that the AIP56 N-terminal metalloprotease can only act when linked to a C-terminal binding domain that, by analogy with other A-B toxins, may assist the protease domain in its membrane translocation into the cytosol
The region described in the publication, spanning residues Cys262 to Cys298, corresponds to region Cys278-Cys314 of the associated UniProt sequence as AIP56 was synthesized as a precursor protein with a cleavable N-terminal signal peptide that was removed during secretion, originating a 497-amino acid mature toxin.
Protein: DP02947 (UniProt A7L035)
Region: residues 22	to 47
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Here we have synthesized peptides corresponding to these regions and analyzed their structures using NMR spectroscopy. The peptide corresponding to the predicted N-terminal amphiphilic helix appears unstructured in aqueous solution.
The locations of Asn21 and Pro22 (corresponding to Asn42 and Pro94 in the full-length protein) are labeled in CfTX-122–47 and CfTX-173–100, respectively, and correspond to the start of the C-terminal tails that are more disordered than the helical regions.
The NMR data for CfTX-122–47 solubilized in water is consistent with the PrDOS results, which indicated that the respective sequence is unstructured.
Secondary α-proton shifts for CfTX-122–47 in H2O (black bars) and in 100 mM SDS (grey bars with black border). Secondary shift values of CfTX-122–47 in an aqueous environment are generally within ±0.1, indicating lack of structure; whereas the values of CfTX-122–47 in SDS are lower than −0.1 ppm between residues 4 and 19 (corresponding to residues 25 and 40 in the full-length protein), indicating an α-helical structure in this environment.
The region predicted to form an amphiphilic α-helix in CfTX-1 (residues 25–32) appears to be disordered in aqueous solution based on the chemical shift analysis of CfTX-122–47.
Protein: DP02947 (UniProt A7L035)
Region: residues 22	to 47
Annotation: disorder to order (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Secondary α-proton shifts for CfTX-122–47 in H2O (black bars) and in 100 mM SDS (grey bars with black border). Secondary shift values of CfTX-122–47 in an aqueous environment are generally within ±0.1, indicating lack of structure; whereas the values of CfTX-122–47 in SDS are lower than −0.1 ppm between residues 4 and 19 (corresponding to residues 25 and 40 in the full-length protein), indicating an α-helical structure in this environment.
By contrast, the chemical shift analysis in the presence of 100 mM SDS has several consecutive negative shifts, consistent with helical structure. This helical structure was confirmed by determination of the three-dimensional structure (Figure 2).
The transition of unstructured to helical structure in the presence of membranes or membrane-mimicking environments has previously been shown for other peptides, such as the antimicrobial peptides MSI-78 and magainin-2
Protein: DP02948 (UniProt J7JU64)
Region: residues 29	to 68
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The 1D 1H NMR spectra of αB-VxXXIVA isomers in phosphate buffer at pH 5.8 show that the majority of the amide protons fall within the 8.0–9.0 ppm range (Fig. 6); the lack of chemical shift dispersion here and elsewhere in the spectrum indicates that these isomers lack any significant tertiary structure. The same was true at pH 7.0 (Fig. S1, S2, S3). NMR spectra were also acquired in the presence of 3–10 mM CaCl2 to ascertain whether calcium had any effect on their conformation, but no change in chemical shift dispersion was observed
Protein: DP02948 (UniProt J7JU64)
Region: residues 29	to 68
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
CD spectra were acquired on all threeαB-VxXXIVA isomers in phosphate buffer. All peptide isomers exhibited minima at around 200 nm (Fig. 7A), indicative of a random coil conformation with no α-helical and β-sheet content, and consistent with our NMR results.
Protein: DP02949 (UniProt O25555)
Region: residues 96	to 125
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
However, another CD analysis (supplemental Fig. S6C) indicated that the 30-residue HP0895Ctp is unstructured.
Protein: DP02949 (UniProt O25555)
Region: residues 96	to 125
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
In contrast, the unstructured C-terminal region of HP0895 was responsible for binding to HP0894 and underwent a conformational change in the process.
Based on these results, and to confirm the binding of this C-terminal region of HP0895 with HP0894, we synthesized a 30-residue peptide, HP0895Ctp (Val96–Ser125), corresponding to the C-terminal region of HP0895 and investigated the interaction between this peptide and HP0894. A series of two-dimensional [1H-15N] TROSY spectra of 15N-labeled HP0894 was recorded with successive additions of unlabeled HP0895Ctp (0, 0.5, 1, and 2 molar equivalents). As shown in Fig. 2C, obvious chemical shift changes in the slow exchange mode on the NMR time scale were observed for a lot of the residues of HP0894, and some other residues showed chemical shift changes in fast exchange mode. Chemical shift changes from the residues in the slow exchange mode were completed with equimolar HP0895Ctp, whereas fast exchange mode chemical shift changes continued above 1:1 molar ratio in an HP0895Ctp concentration-dependent manner. These results indicate that the dissociation constants (Kd) of HP0895Ctp (and consequently HP0895) with HP0894 are relatively small (∼10−8 m or smaller) (27). Thus, HP0894-HP0895 binding is strong. In addition, these results confirmed that HP0895 binds to HP0894 via its C-terminal region with a binding stoichiometry of 1:1.
Our results show that the cellular level functioning of HP0894 is inactivated by binding with HP0895. The direct interaction of these two proteins occurs mainly between the 30-residue C-terminal tail of HP0895 and the N-terminal secondary structure elements and an adjacent C-terminal β-strand of HP0894. Upon binding to HP0894, that unstructured C terminus tail of HP0895 seems to be transformed into structured form.
Taken together, it is concluded that the HP0894-HP0895 protein couple is a TA system in H. pylori, where HP0894 is a toxin with an RNase function, whereas HP0895 is an antitoxin functioning by binding to both the toxin and DNA.
Protein: DP02949 (UniProt O25555)
Region: residues 96	to 125
Annotation: disorder to order (Structural transition)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
In contrast, the unstructured C-terminal region of HP0895 was responsible for binding to HP0894 and underwent a conformational change in the process.
Thus, the unstructured HP0895 C-terminal region transforms into a structured form upon binding to HP0894. Furthermore, far-UV CD titrations of HP0894 with HP0895ctp yield a 1:1 stoichiometry with Kd of ∼10−8 M
Our results show that the cellular level functioning of HP0894 is inactivated by binding with HP0895. The direct interaction of these two proteins occurs mainly between the 30-residue C-terminal tail of HP0895 and the N-terminal secondary structure elements and an adjacent C-terminal β-strand of HP0894. Upon binding to HP0894, that unstructured C terminus tail of HP0895 seems to be transformed into structured form.
Protein: DP02949 (UniProt O25555)
Region: residues 96	to 125
Annotation: toxin sequestering activity (Molecular function)
Evidence: experimental phenotypic evidence used in manual assertion
Supporting statements:
Taken together, it is concluded that the HP0894-HP0895 protein couple is a TA system in H. pylori, where HP0894 is a toxin with an RNase function, whereas HP0895 is an antitoxin functioning by binding to both the toxin and DNA.
HP0894 has RNase activity on mRNA, and HP0895 inhibits the decay of mRNA by HP0894. Moreover, HP0894 expression has a toxic effect on E. coli cell growth, but the co-expression of HP0895 neutralizes that HP0894 toxicity.
Our results show that the cellular level functioning of HP0894 is inactivated by binding with HP0895. The direct interaction of these two proteins occurs mainly between the 30-residue C-terminal tail of HP0895 and the N-terminal secondary structure elements and an adjacent C-terminal β-strand of HP0894. Upon binding to HP0894, that unstructured C terminus tail of HP0895 seems to be transformed into structured form.
Protein: DP02950 (UniProt P0C079)
Region: residues 43	to 65
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The following part (Arg43 to Arg65) is largely unstructured as evidenced by random CSI values and low heteronuclei (1H–15N) nuclear Overhauser enhancement (NOE) values (Fig. S2d and e).
Protein: DP02950 (UniProt P0C079)
Region: residues 43	to 79
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
In summary, antitoxin RelB possesses a well-folded core domain (Met1–Glu42) at its N-terminus followed by a flexible region (Arg43–Leu79) at its C-terminus, a modular pattern typical of many other antitoxins.
Protein: DP02950 (UniProt P0C079)
Region: residues 52	to 79
Annotation: protein binding (Molecular function)
Evidence: experimental phenotypic evidence used in manual assertion
Supporting statements:
To determine the region within RelB that is responsible for RelB−RelE interaction and antitoxic activity, full-length RelB (RelB1-79) and two N-terminus-truncated antitoxin sequences (RelB36-79 and RelB52-79) were coexpressed with RelE toxin in a single operon using the pBAD promoter control. E. coli strain TOP10 cells, carrying the plasmids, were grown in LB medium, and 0.2% arabinose was added at time zero. The growth rate observed for RelB36-79:RelE- or RelB52-79:RelE-expressing bacteria was indifferent from that of the full-length-RelB:RelE-expressing bacteria (Figure 6A). As a positive control, expression of the RelE toxin using the same pBAD system led to significant growth arrest. The result suggests that RelB interacts and neutralizes RelE toxicity via its C-terminal region. The exact binding domain should be therefore located in the last 28 residues of RelB.
We showed evidence that (i) RelB possesses a high content of α-helical and flexible regions and exhibits a conformationally induced fit upon toxin binding, as many proteins featuring disordered regions do, (ii) RelE is a well-folded protein, displaying a high content of antiparallel β-sheets and β-turns, (iii) RelB, RelE, and the RelBE complex display considerably different thermodynamic stabilities that most likely serve as an important functional basis that regulates the activity of the system under different conditions, (iv) the C-terminal part of RelB is responsible for RelB−RelE interaction, and (iv) the C-terminal part of RelB is likely to be unfolded due to high sensitivity to protease activity and this part interacts with RelE to become more resistant to degradation.
Protein: DP02950 (UniProt P0C079)
Region: residues 52	to 79
Annotation: toxin sequestering activity (Molecular function)
Evidence: experimental phenotypic evidence used in manual assertion
Supporting statements:
The RelE and RelB proteins constitute the RNA interferase (toxin) and its cognate inhibitor (antitoxin) components of the Escherichia coli relBE toxin−antitoxin system.
To determine the region within RelB that is responsible for RelB−RelE interaction and antitoxic activity, full-length RelB (RelB1-79) and two N-terminus-truncated antitoxin sequences (RelB36-79 and RelB52-79) were coexpressed with RelE toxin in a single operon using the pBAD promoter control. E. coli strain TOP10 cells, carrying the plasmids, were grown in LB medium, and 0.2% arabinose was added at time zero. The growth rate observed for RelB36-79:RelE- or RelB52-79:RelE-expressing bacteria was indifferent from that of the full-length-RelB:RelE-expressing bacteria (Figure 6A). As a positive control, expression of the RelE toxin using the same pBAD system led to significant growth arrest. The result suggests that RelB interacts and neutralizes RelE toxicity via its C-terminal region. The exact binding domain should be therefore located in the last 28 residues of RelB.
Protein: DP02950 (UniProt P0C079)
Region: residues 47	to 79
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
A 1H-15N HSQC spectrum of 15N,13C-labeled RelBC alone displays poor dispersion of NH resonance (7.9-8.5 ppm), indicating that this C-terminal region of RelB is largely unstructured in its free state (Fig. 2B).
Protein: DP02950 (UniProt P0C079)
Region: residues 47	to 79
Annotation: disorder to order (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Titration of labeled RelBC with unlabeled RelER81A/R83A shows dramatic chemical shift changes in a similar slow exchange regime (Fig. 2, B and D). The well dispersed spectrum of RelBC in the bound state suggests that RelER81A/R83A binding induces the folding of RelBC.
The chemical shift index analysis (30) of RelBC peptide in both free and bound states revealed a disordered-to-ordered conformation change upon the complex formation (supplemental Fig. S3, A and B).
Cross-references: PDB:2KC8
Protein: DP02950 (UniProt P0C079)
Region: residues 47	to 79
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Titration of labeled RelBC with unlabeled RelER81A/R83A shows dramatic chemical shift changes in a similar slow exchange regime (Fig. 2, B and D). The well dispersed spectrum of RelBC in the bound state suggests that RelER81A/R83A binding induces the folding of RelBC.
The chemical shift index analysis (30) of RelBC peptide in both free and bound states revealed a disordered-to-ordered conformation change upon the complex formation (supplemental Fig. S3, A and B).
Cross-references: PDB:2KC8
Protein: DP02950 (UniProt P0C079)
Region: residues 47	to 79
Annotation: toxin sequestering activity (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The residual activity of RelER81A/R83A is completely abolished by the addition of the C-terminal domain of RelB antitoxin, RelBC (residues Lys47 to Leu79) (Fig. 1, A-C).
Our structures indicate that RelB counteracts the toxic activity of RelE by displacing alpha4 helix from the catalytically competent position found in the free RelE structure.
Cross-references: PDB:2KC8
Protein: DP02950 (UniProt P0C079)
Region: residues 47	to 79
Annotation: disorder (Structural state)
Evidence: temperature-induced protein unfolding evidence used in manual assertion
Supporting statements:
The apparent melting temperature of RelBC was estimated to be below 10 °C (Fig. 1b). In summary, antitoxin RelB possesses a well-folded core domain (Met1–Glu42) at its N-terminus followed by a flexible region (Arg43–Leu79) at its C-terminus, a modular pattern typical of many other antitoxins.
Protein: DP02950 (UniProt P0C079)
Region: residues 47	to 79
Annotation: disorder (Structural state)
Evidence: chromatography evidence used in manual assertion
Supporting statements:
Firstly, size-exclusion analysis of RelB (9.38 kDa) yielded an apparent molecular mass (66.9 kDa) that is significantly larger than that of a dimer (as exhibited by many other antitoxin dimers) and closer to that of a septamer. Since the determination of molecular mass by SEC is related to the shape of the molecule, it is less accurate with nonglobular proteins. It is likely that the SEC method overestimates the size of RelB because the unstructured C-terminal region does not fold into a compact globular conformation.
Protein: DP02951 (UniProt P01501)
Region: residues 44	to 70
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Analysis of chemical shifts and steady state heteronuclear nuclear Overhauser effect (NOE) patterns indicated that melittin is unstructured without TFE, but assumes stable helical conformation above a certain TFE concentration threshold.
The 15N-HSQC spectrum of melittin in aqueous solution at pH 7.0 without TFE (Figure 2B) showed a limited chemical shift dispersion along the 1H dimension indicative of an unstructured form.
It is known that melittin is unstructured at low concentrations in aqueous solutions, and more structured when bound to lipids, cell-surface GAGs, and proteins.
Protein: DP02951 (UniProt P01501)
Region: residues 44	to 70
Annotation: disorder to order (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
At 30% TFE (Figure 4B), there is an overall reduction in flexibility manifested by the higher positive heteronuclear NOEs. In general, the trans conformer was less flexible than the cis, and the difference is more pronounced for residues 15-17, and 19-21. The trans conformer has large positive heteronuclear NOEs (0.57 - 0.94 for residues 4-24) throughout the chain, and this is consistent with the formation of stable helices.
Protein: DP02951 (UniProt P01501)
Region: residues 58	to 63
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
We identified the amino acids in melittinimportant for binding to HAA by saturation-transfer difference (STD) nuclearmagnetic resonance (NMR) experiments, and analysis of NMR line broadening upon titration of melittin with HAA. Our results suggest that hydrophobic resi-dues Ile17 and Ile20 on the C-terminal region of melittin are in close contactwith HAA in the melittin-HAA complex.
The melittin residues that showed the highest signal broadening upon addition of HAA were Ile17, and Ile20, with above 60% decrease in peak intensity (Figure 2). Next to these are Leu13, Ala15, Leu16, Ser18, and Trp19, which showed at least 50% decrease in peak amplitude. It is important to note that most of these residues have hydrophobic side chains, thus suggesting that hydrophobic interaction between HAA and melittin drives the binding event, consistent with our ITC findings.