LiteFold/DisProt huggingface dataset:
Protein: DP00246 (UniProt P21758)
Region: residues 1	to 50
Annotation: protein binding (Molecular function)
Evidence: affinity chromatography evidence used in manual assertion
Supporting statements:
Although non-specific binding proteins, which were also detected when using the control resin, appeared on the SDS– PAGE, the specifically binding proteins (shown by the arrows in Fig. 6) were eluted from the gel. And then, the partial amino acid sequences of these proteins were determined as shown in Table 1 (by APRO Life Science Institute).
Protein: DP00246 (UniProt P21758)
Region: residues 1	to 50
Annotation: protein binding (Molecular function)
Evidence: glutathione S-transferase pull-down assay evidence used in manual assertion
Supporting statements:
The results of Western blotting shown in Fig. 7 proved that all HSP90, HSP70 and
GAPDH could interact with the cytoplasmic domain of MSR.
Protein: DP00246 (UniProt P21758)
Region: residues 1	to 50
Annotation: heat shock protein binding (Molecular function)
Evidence: affinity chromatography evidence used in manual assertion
Supporting statements:
Although non-specific binding proteins, which were also detected when using the control resin, appeared on the SDS– PAGE, the specifically binding proteins (shown by the arrows in Fig. 6) were eluted from the gel. And then, the partial amino acid sequences of these proteins were determined as shown in Table 1 (by APRO Life Science Institute). Based on these data, the five known and one unknown protein were identified as the binding proteins (Table 1). Of these proteins, HSP90, HSP70 and GAPDH were the focus of attention for their possible participation in a receptor function.
Protein: DP00246 (UniProt P21758)
Region: residues 1	to 50
Annotation: protein binding (Molecular function)
Evidence: co-immunoprecipitation evidence used in manual assertion
Supporting statements:
Following this result, immunoprecipitation assays were performed to investigate whether these interactions occur in the cells or not. The results showed that all three kinds of protein were precipitated with MSR (Fig. 8). This experiments confirmed that HSP90, HSP70 and GAPDH directly associate with the cytoplasmic domain of MSR in vitro and in vivo.
Protein: DP00250 (UniProt P01555)
Region: residues 44	to 54
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
In this study, we present crystal structures of the intrinsically active CTY30S mutant, providing the first structural views of an active form of CT or LT. To enable accurate structureactivity comparisons, we also independently determined the structure of wild-type CT at higher resolution and accuracy than were previously available (10). Our six new CT structures show an A2 tail conformation that differs markedly from that found in the original CT structure, with interesting implications for toxicity. In addition, the differences between CTY30S and wild-type CT structures permit further insight into the mechanism of CT/LT activation
Residues in the 25-36 “activation loop” were ordered in the originally determined wild-type CT structure and in wild-type LT structures but displayed mostly higher than average B-factors in wild-type LT structures. These residues were included in the molecular replacement model used for CTY30S structure determinations, but density in the CTY30S structures did not support the inclusion of activation loop residues in the final models, suggesting that the activation loop is disordered in CTY30S (Figure 2).
The only major structural changes observed between wildtype CT and CTY30S occur in the level of flexibility found in the activation and active site loops



B-Factors along the activation loop are higher than the average B-factors for the A1 subunits of the CT/LT structures, suggesting that this loop already has a tendency toward disorder (Figure 5). Missing density in the wild-type CT form 2 structure for residues 33-35 further underscores the high level of thermal motion naturally present in this loop (Figure 2). 
The 'activated' state where disorder is observed corresponds to the Y30S point mutant, which is shown to have hogh enzymatic activity, unlike wtCT
Numbering needs to be shifted by 18 residues to match the Uniprot sequence due to the presence of the signal peptide. 
Cross-references: PDB:1S5B:A
PDB:1S5C:A
Protein: DP00250 (UniProt P01555)
Region: residues 44	to 54
Annotation: order to disorder (Structural transition)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
In this study, we present crystal structures of the intrinsically active CTY30S mutant, providing the first structural views of an active form of CT or LT. To enable accurate structureactivity comparisons, we also independently determined the structure of wild-type CT at higher resolution and accuracy than were previously available (10). Our six new CT structures show an A2 tail conformation that differs markedly from that found in the original CT structure, with interesting implications for toxicity. In addition, the differences between CTY30S and wild-type CT structures permit further insight into the mechanism of CT/LT activation
The fact that the activation loop is consistently disordered in CTY30S structures but is ordered in wild-type CT structures, despite different packing environments among the crystal forms, indicates that the activation loop is not likely to be affected by crystal packing interactions in these structures. In all wild-type CT structures, both the 25-36 activation loop and 47-56 active site loop are ordered, with a minor exception of the activation loop of wild-type CT form 2. 
The only major structural changes observed between wildtype CT and CTY30S occur in the level of flexibility found in the activation and active site loops
Role of the ActiVation Loop in the ActiVation Mechanism. Activation loop disorder is the only major conformational difference between the high-resolution wild-type CT structures and all the CTY30S structures. This feature is thus likely to be a key factor for the difference in intrinsic activity between wild-type CT and constitutively active CTY30S, suggesting that disorder in this loop is necessary for the transition between proenzyme and active enzyme


Cross-references: PDB:1S5F
PDB:1S5E
Protein: DP00250 (UniProt P01555)
Region: residues 19	to 212
Annotation: disorder (Structural state)
Evidence: cleavage assay evidence used in manual assertion
Supporting statements:
the isolated CTA1 polypeptide has a disordered conformation with minimal enzymatic activity against Gs or synthetic substrates at 37°C (Murayama et al., 1993; Ray et al., 2012). When CTA1-ARF interactions have been examined at 37°C, CTA1 and ARF are typically added together before incubation at 37°C (Murayama et al., 1993; Pande et al., 2007). This protocol allows ARF6 to stabilize the folded conformation of CTA1 initially present at low temperature, but it does not determine whether ARF6 can activate the disordered CTA1 subunit. In this paper we examined whether ARF6 can activate a disordered, as well as a structured, CTA1 polypeptide. A biochemical and biophysical analysis of CTA1 structure / function indicated that ARF6 cannot promote the refolding of disordered CTA1 to an active state. Instead, lipid rafts shifted the disordered, 37°C structure of CTA1 to a folded conformation with a basal level of activity that could be further stimulated by ARF6.
CTA1-ARF6 interactions were initially examined with a protease sensitivity assay (Fig. 1). The isolated CTA1 polypeptide shifts from a protease-resistant conformation to a protease sensitive conformation with increasing temperature, which is consistent with its intrinsic thermal instability (Pande et al., 2007).
Because free CTA1 is an unstable protein, the translocated pool of toxin would remain in a disordered state at 37°C and must therefore engage components of the host cell to regain an ordered, active conformation
Protein: DP00250 (UniProt P01555)
Region: residues 19	to 212
Annotation: disorder to order (Structural transition)
Evidence: cleavage assay evidence used in manual assertion
Supporting statements:
the isolated CTA1 polypeptide has a disordered conformation with minimal enzymatic activity against Gs or synthetic substrates at 37°C (Murayama et al., 1993; Ray et al., 2012). When CTA1-ARF interactions have been examined at 37°C, CTA1 and ARF are typically added together before incubation at 37°C (Murayama et al., 1993; Pande et al., 2007). This protocol allows ARF6 to stabilize the folded conformation of CTA1 initially present at low temperature, but it does not determine whether ARF6 can activate the disordered CTA1 subunit. In this paper we examined whether ARF6 can activate a disordered, as well as a structured, CTA1 polypeptide. A biochemical and biophysical analysis of CTA1 structure / function indicated that ARF6 cannot promote the refolding of disordered CTA1 to an active state. Instead, lipid rafts shifted the disordered, 37°C structure of CTA1 to a folded conformation with a basal level of activity that could be further stimulated by ARF6.
ARF6/GTP but not ARF6/GDP prevented the temperature-induced transition of CTA1 to a protease-sensitive conformation (Fig. 1A). This confirmed our previous report (Pande et al., 2007) and demonstrated the active form of ARF6 can stabilize the folded, protease-resistant conformation of CTA1
we heated CTA1 to 37°C before the addition of ARF6. Under this condition, neither ARF6/GDP nor ARF6/GTP protected CTA1 from proteolysis.
In contrast to ARF6, large unilamellar vesicles (LUVs) mimicking the composition of a lipid raft could protect CTA1 from proteolysis even when they were added after toxin unfolding had already occurred (Fig. 1B). This was consistent with previous biophysical measurements demonstrating the chaperone-like ability of lipid rafts to stabilize folded CTA1 and to induce both a gain-of-structure and gain-of-function in the disordered CTA1 polypeptide (Ray et al., 2012). ARF6 and lipid rafts thus exhibited overlapping yet distinct interactions with CTA1: both could prevent the thermal unfolding of CTA1 to a protease-sensitive conformation, but only lipid rafts could shift disordered CTA1 back to a protease-resistant structure
Because CTA1 is disordered at 37°C and ARF6/GTP does not promote the refolding of disordered CTA1 (Fig. 2, Table 1), it is necessary for other host factors such as lipid rafts to participate in shifting CTA1 from a disordered conformation to a partially folded active state that is competent for functional interaction with ARFs. The gain-of-structure resulting from toxin-raft interactions allows ARF6/GTP to induce an additional conformational change (Fig. 4, Table 1) that presumably stabilizes displacement of the occluding CTA1 “activation loop” and consequently optimizes the enzymatic activity of CTA1 (Fig. 5A).
Protein: DP00250 (UniProt P01555)
Region: residues 19	to 212
Annotation: protein binding (Molecular function)
Evidence: cleavage assay evidence used in manual assertion
Supporting statements:
the isolated CTA1 polypeptide has a disordered conformation with minimal enzymatic activity against Gs or synthetic substrates at 37°C (Murayama et al., 1993; Ray et al., 2012). When CTA1-ARF interactions have been examined at 37°C, CTA1 and ARF are typically added together before incubation at 37°C (Murayama et al., 1993; Pande et al., 2007). This protocol allows ARF6 to stabilize the folded conformation of CTA1 initially present at low temperature, but it does not determine whether ARF6 can activate the disordered CTA1 subunit. In this paper we examined whether ARF6 can activate a disordered, as well as a structured, CTA1 polypeptide. A biochemical and biophysical analysis of CTA1 structure / function indicated that ARF6 cannot promote the refolding of disordered CTA1 to an active state. Instead, lipid rafts shifted the disordered, 37°C structure of CTA1 to a folded conformation with a basal level of activity that could be further stimulated by ARF6.
ARF6/GTP but not ARF6/GDP prevented the temperature-induced transition of CTA1 to a protease-sensitive conformation (Fig. 1A). This confirmed our previous report (Pande et al., 2007) and demonstrated the active form of ARF6 can stabilize the folded, protease-resistant conformation of CTA1
In contrast to ARF6, large unilamellar vesicles (LUVs) mimicking the composition of a lipid raft could protect CTA1 from proteolysis even when they were added after toxin unfolding had already occurred (Fig. 1B). 
Because CTA1 is disordered at 37°C and ARF6/GTP does not promote the refolding of disordered CTA1 (Fig. 2, Table 1), it is necessary for other host factors such as lipid rafts to participate in shifting CTA1 from a disordered conformation to a partially folded active state that is competent for functional interaction with ARFs. The gain-of-structure resulting from toxin-raft interactions allows ARF6/GTP to induce an additional conformational change (Fig. 4, Table 1) that presumably stabilizes displacement of the occluding CTA1 “activation loop” and consequently optimizes the enzymatic activity of CTA1 (Fig. 5A).
Protein: DP00250 (UniProt P01555)
Region: residues 19	to 212
Annotation: disorder (Structural state)
Evidence: Fourier-transform infrared spectroscopy evidence used in manual assertion
Supporting statements:
the isolated CTA1 polypeptide has a disordered conformation with minimal enzymatic activity against Gs or synthetic substrates at 37°C (Murayama et al., 1993; Ray et al., 2012). When CTA1-ARF interactions have been examined at 37°C, CTA1 and ARF are typically added together before incubation at 37°C (Murayama et al., 1993; Pande et al., 2007). This protocol allows ARF6 to stabilize the folded conformation of CTA1 initially present at low temperature, but it does not determine whether ARF6 can activate the disordered CTA1 subunit. In this paper we examined whether ARF6 can activate a disordered, as well as a structured, CTA1 polypeptide. A biochemical and biophysical analysis of CTA1 structure / function indicated that ARF6 cannot promote the refolding of disordered CTA1 to an active state. Instead, lipid rafts shifted the disordered, 37°C structure of CTA1 to a folded conformation with a basal level of activity that could be further stimulated by ARF6.
CTA1 is in a folded conformation at low temperatures (Pande et al., 2007). We therefore used the FTIR spectrum of CTA1 at 10°C as a reference for the structure of folded CTA1 (Fig. 2A). Analysis of the 10°C FTIR spectrum from CTA1 indicated that the folded toxin contains 35 ± 4% α-helical and 49 ± 3% β-sheet content (Table 1).
Due to its intrinsic thermal instability, heating CTA1 to 37°C resulted in a loss of both α-helical and βsheet structures (Fig. 2B). Concomitant with this loss of secondary structure, the percentage of CTA1 irregular structure shifted from 10 ± 3% at 10°C to 55 ± 2% at 37°C (Table 1). 
Because free CTA1 is an unstable protein, the translocated pool of toxin would remain in a disordered state at 37°C and must therefore engage components of the host cell to regain an ordered, active conformation.
Protein: DP00250 (UniProt P01555)
Region: residues 19	to 212
Annotation: protein binding (Molecular function)
Evidence: Fourier-transform infrared spectroscopy evidence used in manual assertion
Supporting statements:
the isolated CTA1 polypeptide has a disordered conformation with minimal enzymatic activity against Gs or synthetic substrates at 37°C (Murayama et al., 1993; Ray et al., 2012). When CTA1-ARF interactions have been examined at 37°C, CTA1 and ARF are typically added together before incubation at 37°C (Murayama et al., 1993; Pande et al., 2007). This protocol allows ARF6 to stabilize the folded conformation of CTA1 initially present at low temperature, but it does not determine whether ARF6 can activate the disordered CTA1 subunit. In this paper we examined whether ARF6 can activate a disordered, as well as a structured, CTA1 polypeptide. A biochemical and biophysical analysis of CTA1 structure / function indicated that ARF6 cannot promote the refolding of disordered CTA1 to an active state. Instead, lipid rafts shifted the disordered, 37°C structure of CTA1 to a folded conformation with a basal level of activity that could be further stimulated by ARF6.
When ARF6/GTP was added to CTA1 before heating to 37°C, the toxin retained its full αhelical structure and a substantial amount of its β-sheet structure at 37°C (Fig. 2C, Table 1). 
The lipochaperone function of lipid rafts (Ray et al., 2012) could act in conjunction with ARF to place disordered CTA1 in a folded, active conformation. This possibility was examined by FTIR spectroscopy (Fig. 4). CTA1 was heated to 37°C for 15 minutes before exposure to lipid raft-mimicking LUVs. Measurements of toxin structure were then recorded after an additional 30 minutes at 37°C (Fig. 4A). In the presence of lipid raft LUVs, the disordered conformation of CTA1 gained both α-helical and β-sheet content (Table 1). An additional gain of β-sheet structure and a further loss of irregular structure were detected when the disordered CTA1 subunit was incubated with both lipid rafts and ARF6/GTP (Fig. 4B). Under this condition, CTA1 exhibited an ordered conformation that matched the structural content of the folded, 10°C toxin (Table 1)
Because CTA1 is disordered at 37°C and ARF6/GTP does not promote the refolding of disordered CTA1 (Fig. 2, Table 1), it is necessary for other host factors such as lipid rafts to participate in shifting CTA1 from a disordered conformation to a partially folded active state that is competent for functional interaction with ARFs. The gain-of-structure resulting from toxin-raft interactions allows ARF6/GTP to induce an additional conformational change (Fig. 4, Table 1) that presumably stabilizes displacement of the occluding CTA1 “activation loop” and consequently optimizes the enzymatic activity of CTA1 (Fig. 5A)
Protein: DP00250 (UniProt P01555)
Region: residues 44	to 54
Annotation: disorder to order (Structural transition)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
The CTA1 activation loop (residues 25 to 40), earlier suggested to be important in CT and LT activation (24), interacts with both partners but in different ways. The loop forms an ordered coil when bound by CTA2 in wildtype holo-CT structures, but residues 25 to 33 rearrange to form an amphipathic helix upon binding ARF6-GTP (Figs. 1 and 3).
Interestingly, disorder of activation-loop residues 26 to 36 in a CTA1:Y30S mutant results in a holotoxin variant with intrinsic activity (3). Here, activation of CTA1 by ARF6-GTP leads to a well-structured activation loop.
Cross-references: PDB:2A5F
PDB:2A5D
Protein: DP00250 (UniProt P01555)
Region: residues 44	to 54
Annotation: protein binding (Molecular function)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
The CTA1 activation loop (residues 25 to 40), earlier suggested to be important in CT and LT activation (24), interacts with both partners but in different ways. The loop forms an ordered coil when bound by CTA2 in wildtype holo-CT structures, but residues 25 to 33 rearrange to form an amphipathic helix upon binding ARF6-GTP (Figs. 1 and 3).
Interestingly, disorder of activation-loop residues 26 to 36 in a CTA1:Y30S mutant results in a holotoxin variant with intrinsic activity (3). Here, activation of CTA1 by ARF6-GTP leads to a well-structured activation loop.
Cross-references: PDB:2A5F
PDB:2A5D
Protein: DP00250 (UniProt P01555)
Region: residues 186	to 204
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
We report the first NMR based solution structural data for the Cholera Toxin enzymatic domain (CTA1). We show that this free enzymatic domain partially unfolds at the C-terminus, and binds its protein partners at both the beginning and end of this activation process.
To evaluate whether the unfolded regions detected within full-length CTA1 are localized to the carboxyl-terminus, we generated and analyzed a mutant form of CTA1 without the carboxyl-terminal region, called CTA1-T2 (N1-F167). For CTA1- T2, the sequence was truncated between F167↓P168P169 as P168P169 make extensive contacts with the CTA2 linker in the holotoxin crystal structure (10) and are likely to destabilize the free enzymatic domain. Spectra of the refolded CTA1-T2 sample show similar chemical shift (δ) values and dispersion to the CTA1 sample (Supplementary Figure 4). Most of the changes in the 1H/15N HSQC spectrum corresponded to a decrease in the number of resonances within the random coil region, while leaving unaffected the dispersed resonances, suggesting that the structure of the catalytic core domain was unaffected by the truncation of the Cterminus.
The 2D NMR 1H/15N HSQC spectrum of recombinant CTA1 (N1-G186) (Figure 3a) revealed that CTA1 is partially unfolded and displays the characteristics of a molten globule in solution [for review, see (34)]. Notably, only 20% of the total amide protons resonated either up-field or down-field when compared to the random coil chemical shift region between 7.5 and 8.5 ppm (34–36). Moreover, there is considerable variation in peak intensity within the 1H/15N HSQC spectrum, likely due to intermediate exchange broadening.
The free CTA1 enzymatic domain is partially unfolded, in contrast to when it is complexed with either CTA2 in the holotoxin or with ARF6-GTP following retrotranslocation. Further strengthening this argument was that the refolded recombinant CTA1 is catalytically active (23) and, due to its thermal instability, CTA1 is a substrate for export to the cytosol via the ERAD pathway (33).
Numbering needs to be shifted by 18 residues to match the Uniprot sequence
Cross-references: BMRB:15162
Protein: DP00250 (UniProt P01555)
Region: residues 186	to 204
Annotation: disorder to order (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
We report the first NMR based solution structural data for the Cholera Toxin enzymatic domain (CTA1). We show that this free enzymatic domain partially unfolds at the C-terminus, and binds its protein partners at both the beginning and end of this activation process.
Many of the resonances present in the central random-coil region of the free enzyme are dispersed in the NMR spectrum for the active complex sample where CTA1 is selectively labeled. Thus, the binding of ARF6-GTP ligand to CTA1 results in an ordering (folding) of partially unfolded regions within the free enzymatic domain, indicating an allosteric coupling between these interaction partners (37).
The free CTA1 enzymatic domain is partially unfolded, in contrast to when it is complexed with either CTA2 in the holotoxin or with ARF6-GTP following retrotranslocation. Further strengthening this argument was that the refolded recombinant CTA1 is catalytically active (23) and, due to its thermal instability, CTA1 is a substrate for export to the cytosol via the ERAD pathway (33).
We propose that the primary role of ARF6- GTP binding is in site-to-site allostery (37) that couples the refolding of the C-terminus of CTA1 with the formation of a high-affinity CTA1:ARF6-GTP complex
Thus, CTA1 undergoes an ordered (holotoxin) to disordered (free CTA1) to ordered (CTA1:ARF6-GTP complex) structural transition (Figure 7) to accommodate the dual requirements of retrotranslocation and activation. This model proposes that the partially unfolded free enzyme recruits proteins from the ERAD pathway to promote its own retrotranslocation from the ER to the cytosol.
Numbering needs to be shifted by 18 residues to match the Uniprot sequence
Cross-references: BMRB:15162