LiteFold/DisProt huggingface dataset:
Protein: DP00250 (UniProt P01555)
Region: residues 186	to 204
Annotation: protein binding (Molecular function)
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.
Cross-references: BMRB:15162
Protein: DP00250 (UniProt P01555)
Region: residues 186	to 204
Annotation: disorder to order (Structural transition)
Evidence: fluorescence 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.
Partial unfolding of CTA1 is further illustrated by using bis-ANS fluorescence as an indicator of the exposed hydrophobic character of the free enzyme, which is substantially reduced when bound to ARF6-GTP
The bis-ANS fluorescence reflects the extent of hydrophobic exposure (38) of CTA1 when compared to both the CTA1:ARF6-GTP complex and Gdm-HCl denatured samples (Figure 4). These data indicate bis-ANS fluorescence of the full length CTA1 sample is reduced in the truncated CTA1-T2 sample, and further reduced in the CTA1:ARF6-GTP sample or in the presence of chemical denaturants. These results indicate a loss of hydrophobic bis-ANS binding sites upon ARF6-GTP binding to the enzymatic domain, when fully unfolded or when truncated at the C-terminus. Therefore, ARF6-GTP binding is sufficient to induce profound changes in CTA1: from a partially disordered state in the free enzyme to an ordered state within the CTA1:ARF6-GTP complex.
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 reported in the publication needs to be shifted by 18 residues to match the Uniprot sequence due to the presence of a signal peptide
Protein: DP00250 (UniProt P01555)
Region: residues 19	to 212
Annotation: lipid 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.
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 19	to 212
Annotation: lipid 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.
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 186	to 204
Annotation: disorder (Structural state)
Evidence: fluorescence 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. Deviations from random coil chemical shifts (Δδcoil) indicate helix formation in the activation loop, which is essential to open the toxin’s active site, occurs prior to its association with human protein ARF6
Partial unfolding of CTA1 is further illustrated by using bis-ANS fluorescence as an indicator of the exposed hydrophobic character of the free enzyme, which is substantially reduced when bound to ARF6-GTP
The bis-ANS fluorescence reflects the extent of hydrophobic exposure (38) of CTA1 when compared to both the CTA1:ARF6-GTP complex and Gdm-HCl denatured samples (Figure 4). These data indicate bis-ANS fluorescence of the full length CTA1 sample is reduced in the truncated CTA1-T2 sample, and further reduced in the CTA1:ARF6-GTP sample or in the presence of chemical denaturants. These results indicate a loss of hydrophobic bis-ANS binding sites upon ARF6-GTP binding to the enzymatic domain, when fully unfolded or when truncated at the C-terminus. Therefore, ARF6-GTP binding is sufficient to induce profound changes in CTA1: from a partially disordered state in the free enzyme to an ordered state within the CTA1:ARF6-GTP complex.
Numbering reported in the publication needs to be shifted by 18 residues to match the Uniprot sequence due to the presence of a signal peptide
Protein: DP00250 (UniProt P01555)
Region: residues 254	to 258
Annotation: localization (Biological process)
Evidence: co-localization evidence used in manual assertion
Supporting statements:
A cholera toxin mutant (CTX–K63) unable to raise cAMP levels was used to study in Vero cells the retrograde transport of the toxin A subunit (CTXA–K63), which possesses a COOH-terminal KDEL retrieval signal. Microinjected GTP-g-S inhibits the internalization as well as Golgi–ER transport of CTXA–K63. 
IgGs as well as Fab fragments directed against Erd2p, b-COP, or p23, a new member of the p24 protein family, inhibit or block retrograde transport of CTX-A–K63 from the Golgi without affecting its internalization or its transport to the Golgi. Anti-Erd2p antibodies do not affect the binding of CTX-A to Erd2p, but inhibit the CTX-K63–induced translocation of Erd2p and p53.
To analyze for a possible contribution of the cytoplasmically oriented COOH terminus of Erd2p to the sorting and the retrograde transport of the occupied Erd2p we analyzed the effect of antibodies raised against the COOH terminus of Erd2p on the retrograde transport of CTXA–K63. Anti-Erd2p IgGs injected before start of CTX– K63 uptake, did not significantly affect the retrograde transport from the plasma membrane to the Golgi (Table I). However, when the IgGs were injected 10–20 min after start of CTX–K63 uptake, we observed in ~60% of the cells an accumulation of CTX-A–K63 in perinuclear structures and an inhibition of translocation of CTX-A–K63 from the Golgi to the ER (Table I). 
To exclude the possibility that the anti-Erd2p–induced inhibition of retrograde transport of CTX-A–K63 from the Golgi to the ER had resulted from a decreased binding of CTX-A–K63 to Erd2p, we measured the effect of the anti-Erd2p antibodies on the binding of 125I-labeled CTX-A to permeabilized Golgi vesicles from Sf9 cells overexpressing human Erd2p. The antibody binding was performed at pH 7.2, the binding of CTX-A to Erd2p at pH 6. At pH 6 the binding of CTX-A to Erd2p was still sufficiently high (z60% of the binding observed at pH 5, unpublished results). Preincubation of the permeabilized Golgi membranes with anti-Erd2p antibodies did not affect binding of CTX-A to Erd2p (Fig. 9). Binding of CTX-A was specific as CTX-A could be displaced from the membranes by low concentrations of KDEL peptides but not by peptides without a COOH-terminal KDEL sequence (results not shown here). 
This is a true positive KDEL signal in the Eukaryotic Linear Motif Database
Protein: DP00250 (UniProt P01555)
Region: residues 19	to 212
Annotation: protein binding (Molecular function)
Evidence: mass spectrometry evidence used in manual assertion
Supporting statements:
Cholera toxin is assembled from two subunits in the periplasm of Vibrio cholerae and disassembled in the analogous compartment of target cells, the lumen of the endoplasmic reticulum (ER), before a fragment of it, the A1 chain, is transported into the cytosol. We show that protein disulfide isomerase (PDI) in the ER lumen functions to disassemble and unfold the toxin once its A chain has been cleaved. 
To identify the unfolding activity in the ER lumen, we bound the proteins of the lumenal extract to a Q-Sepharose column. Upon elution with a salt gradient, protein fractions were analyzed for their ability to confer trypsin sensitivity to the A and A1 subunits. Addition of fractions 9 or 10 resulted in complete degradation of these toxin polypeptides (Figure 2A, lanes 18 and 20), similar to the effect observed with the unfractionated extract (lane 4). 
Fractions 9 and 10 contained one major band of ap- proximately 60 kDa (Figure 2B, lanes 8 and 9). This band was excised and identified as protein disulfide isomerase (PDI) by mass spectroscopy. Immunoblotting with an antibody directed against PDI confirmed this identifi- cation (Figure 2C, lanes 8 and 9). 
In addition, further purification of the material in fraction 9 on a S-Sepharose column demonstrated perfect cofractionation of PDI and the unfolding activity (Figure 2D). To confirm that the major unfolding activity in the ER lumen can be attributed to PDI, we immunodepleted PDI from the ex- tract (Figure 2E, left panel, lane 2 versus 1). While mock- depleted extract confered trypsin sensitivity to the toxin A and A1 subunits, the PDI-depleted extract was inactive (Figure 2E, right panel, lanes 6 versus 14). 
Our results provide insight into the pathway of cholera toxin from its synthesis in bacteria up to its arrival in the lumen of the ER in mammalian cells, where its A1 fragment is released from the rest of the toxin and unfolded in preparation for its translocation into the cytosol. 
Our results explain why cholera toxin is assembled and folded in the periplasm of bacteria and disassembled and unfolded in the ER, even though both compartments normally support the folding of polypeptides. One decisive factor is that the A subunit needs to be cleaved into the A1 and A2 fragments for unfolding to occur. The periplasm of bacteria seems to lack proteases that cleave the A chain, and in this compartment, the two subunits can therefore assemble and fold into the native holotoxin.  Once in the ER, PDI binds to the A1 chain and causes it to unfold, but it leaves the ring of B chains intact.
Protein: DP00250 (UniProt P01555)
Region: residues 19	to 212
Annotation: protein binding (Molecular function)
Evidence: co-immunoprecipitation evidence used in manual assertion
Supporting statements:
Cholera toxin is assembled from two subunits in the periplasm of Vibrio cholerae and disassembled in the analogous compartment of target cells, the lumen of the endoplasmic reticulum (ER), before a fragment of it, the A1 chain, is transported into the cytosol. We show that protein disulfide isomerase (PDI) in the ER lumen functions to disassemble and unfold the toxin once its A chain has been cleaved. 
Yeast PDI behaved in a similar way as the mammalian enzyme, despite their low sequence similarity (less than 25% identical amino acids). Purified yeast PDI was un- able to unfold the A subunit under oxidizing conditions (Figure 4A, lane 6 versus 5), but was active under reducing conditions (lane 8 versus 7). Similarly, it gave crosslinks to the A subunit only under reducing conditions (Figure 4B, lane 4 versus 3). 
Our results provide insight into the pathway of cholera toxin from its synthesis in bacteria up to its arrival in the lumen of the ER in mammalian cells, where its A1 fragment is released from the rest of the toxin and unfolded in preparation for its translocation into the cytosol. 
Our results explain why cholera toxin is assembled and folded in the periplasm of bacteria and disassembled and unfolded in the ER, even though both compartments normally support the folding of polypeptides. One decisive factor is that the A subunit needs to be cleaved into the A1 and A2 fragments for unfolding to occur. The periplasm of bacteria seems to lack proteases that cleave the A chain, and in this compartment, the two subunits can therefore assemble and fold into the native holotoxin.  Once in the ER, PDI binds to the A1 chain and causes it to unfold, but it leaves the ring of B chains intact.
Protein: DP00250 (UniProt P01555)
Region: residues 101	to 106
Annotation: protein binding (Molecular function)
Evidence: surface plasmon resonance evidence used in manual assertion
Supporting statements:
To determine whether HSC70 binds directly to CTA1, we perfused HSC70 over a CTA1-coated surface plasmon resonance (SPR) sensor at 15 or 37 °C (Fig. 4A). Previous studies have shown by CD, tryptophan fluorescence, and FTIR spectroscopy that CTA1 maintains a folded conformation at low temperature but transitions to a partially unfolded state at the physiological temperature of 37 °C (7, 49). HSC70 could bind to both the folded conformation of CTA1 present at 15 °C and the disordered conformation of CTA1 present at 37 °C. 
Both HSC70 and HSP90 form tight complexes with CTA1, as little dissociation is seen after removal of the chaperone from the perfusion buffer (Fig. 4A) (17, 18). We previously calculated a 7 nM KD affinity between HSP90 and CTA1 at 37 °C (17). HSC70 exhibited a similar 5 nM KD affinity for CTA1 at 37 °C (Fig. 4B). However, host-toxin interactions are not identical for the two chaperones: HSC70 will bind to either folded or disordered CTA1, whereas HSP90 only recognizes disordered CTA1 (18). 
To identify the CTA1-binding site(s) for HSC70, we generated a series of peptides that contained overlapping amino acid sequences from the entire length of the CTA1 subunit. As summarized in Table 1, HSC70 bound two consecutive peptides spanning CTA1 amino acid residues 73–98. These peptides contained a shared YYIYVI sequence that likely represented the core HSC70-binding site. The YYIYVI sequence alone could not be generated as a soluble hexapeptide, but we found a truncated GQTILSGHST peptide that lacked the YYIYVI motif and a mutant GQTILSGHSTYYLYVL peptide with leucine for iso- leucine substitutions in the YYIYVI motif could not bind to HSC70 (Table 1). These results indicated the YYIYVI motif was recognized by HSC70.  A competition assay further established the specificity of HSC70 binding to the YYIYVI sequence (Fig. 6). For this experiment, CTA1 was appended to an SPR sensor slide and exposed to perfusion buffer containing either HSC70 alone or HSC70 in combination with a CTA1-related peptide. HSC70 could bind to CTA1 in the absence of peptide and in the presence of the mutant GQTILSGHSTYYLYVL peptide. However, HSC70 could not bind to CTA1 in the presence of the GQTILSGH- STYYIYVI peptide. These observations confirmed HSC70 specifically recognizes the YYIYVI sequence in CTA1. 
18 residues need to be added to the numbering reported by the authors to match the Uniprot entry. The binding site YYIYVI corresponds to residues 101-106. HSP70 from  commercial origin, Enzo Life Sciences (Farmingdale, NY) corresponds to Bovine HSP70
Protein: DP00250 (UniProt P01555)
Region: residues 19	to 212
Annotation: protein binding (Molecular function)
Evidence: surface plasmon resonance evidence used in manual assertion
Supporting statements:
Hsp90 bound to CTA1 in an ATP-dependent manner that was blocked by geldanamycin (GA), an established Hsp90 inhibitor.
To address this issue, SPR was used to determine whether Hsp90 could directly interact with the isolated CTA1 subunit at physiological temperature (Fig. 1A). Hsp90 binding to CTA1 occurred in an ATP-dependent manner that was blocked by GA. This demonstrated that Hsp90 could bind to CTA1 at 37 °C. Because CTA1 is in an unfolded conformation at 37 °C (22, 24), this also suggested that Hsp90 recognizes an unfolded conformation of CTA1 during the dislocation event
The interaction between Hsp90 and CTA1 was much stronger than the interaction between GRP94 and CTA1: Hsp90 bound to CTA1 with a KD of 7 nM, whereas GRP94 bound to CTA1 with a KD of 292 nM (Fig. 2 and Table 1). Consistent with the dimeric natures of Hsp90 and GRP94 (31), both chaperones bound to CTA1 in a 2:1 ratio of chaperone:toxin (Fig. 2). The specific, high-affinity interaction between Hsp90-ATP and CTA1 indicated that Hsp90 could be involved with the CT intoxication process
HSP90 origin is extracted from PMID: 31221799 to be Human HSP90 (Commercial origin; Biovision)
Protein: DP00251 (UniProt O88339)
Region: residues 144	to 575
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
In the spectra of the fragments lacking the ENTH domains (His6-epsin 1-(144–575) and His6-AP180-(328–896)), the α-helical characteristics were almost completely lost, whereas the content of random structures increased from 56 to 66% for the epsin 1 fragment and from 55 to 84% for the AP180 fragment (Fig. 4,A and B).
Protein: DP00251 (UniProt O88339)
Region: residues 144	to 575
Annotation: disorder (Structural state)
Evidence: gel-filtration evidence used in manual assertion
Supporting statements:
The observed hydrodynamic size is consistent with either an extended rod with a thickness of 1.5–2.0 nm and a length of some 50 nm or, alternatively, a poorly folded polypeptide chain. Taken together, our data obtained from quantitative gel filtration chromatography and ultracentrifugation strongly suggest that the carboxyl-terminal parts of epsin 1 and AP180 are rather extended, with the consequence that they behave like very large proteins on gel filtration columns and like molecular parachutes during ultracentrifugation.