LiteFold/DisProt huggingface dataset:
Protein: DP04219 (UniProt O95433)
Region: residues 162	to 204
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy-based hydrogen-deuterium exchange evidence used in manual assertion
Supporting statements:
The solved solution structure ensemble of Aha128−335 indicates that human Aha1’s N-terminal domain and C-terminal domain are linked through a long unstructured loop (Figure 4a, Supplemental Figure S4), and there are no tight contacts between the two domains.
Figure 4 specifies the boundaries for the C-terminal and the N-terminal region.
Cross-references: PDB:7DMD
Protein: DP04219 (UniProt O95433)
Region: residues 162	to 204
Annotation: flexible linker (Disorder function)
Evidence: nuclear magnetic resonance spectroscopy-based hydrogen-deuterium exchange evidence used in manual assertion
Supporting statements:
The solved solution structure ensemble of Aha128−335 indicates that human Aha1’s N-terminal domain and C-terminal domain are linked through a long unstructured loop (Figure 4a, Supplemental Figure S4), and there are no tight contacts between the two domains.
Figure 4 specifies the boundaries for the C-terminal and the N-terminal region.
Cross-references: PDB:7DMD
Protein: DP04219 (UniProt O95433)
Region: residues 1	to 27
Annotation: flexible N-terminal tail (Disorder function)
Evidence: nuclear magnetic resonance spectroscopy-based hydrogen-deuterium exchange evidence used in manual assertion
Supporting statements:
This analysis revealed that the presence of N-terminal sequence motif spanning M1 to W27 does not modify the global fold of Aha1's N-terminal domain spanning T28 to G162 (Figure 3a). Moreover, The CSI data demonstrated that the N-terminal sequence motif of Aha1 spanning M1 to W27 mainly adopts a random coil conformation (Figure 3a). In support of this finding, compared with the dynamics behavior of the core region, extremely high flexibility was observed for the N-terminal sequence motif of Aha1 spanning M1 to W27 (Figure 3b,c).
Protein: DP04219 (UniProt O95433)
Region: residues 1	to 27
Annotation: protein folding chaperone (Molecular function)
Evidence: thioflavin T based fluorescence spectroscopy evidence
Supporting statements:
After the confirmation of the interaction between human Aha1 and α-synuclein, ThT assay and transmission electron microscopy (TEM) were applied to test the impact of Aha1 on the aggregation process of α-synuclein. Both the ThT data and the TEM results demonstrated that Aha1 presented an inhibition effect on α-synuclein aggregation (Figure 6d,e).
Protein: DP04219 (UniProt O95433)
Region: residues 1	to 27
Annotation: unfolded protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy-based hydrogen-deuterium exchange evidence used in manual assertion
Supporting statements:
In this study, to test if the N-terminal fragment M1-W27 of Aha1 plays a role in the recognition of disordered proteins, intrinsically disordered α-synuclein was selected as a model protein. 1H-15N-HSQC spectra using 15N-labeled Aha11−338, 15N-labeled Aha128−338 and 15N-labeled Aha128−335 without or with the presence of 10-fold molar excess of unlabeled α-synuclein were recorded (Figure 6). According to the acquired 1H-15N-HSQC titration data, both the N-terminal fragment M1-W27 and the C-terminal RLF (R336L337F338) motif of Aha1 contributed to the recognition of α-synuclein.
Protein: DP04219 (UniProt O95433)
Region: residues 1	to 27
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
This analysis revealed that the presence of N-terminal sequence motif spanning M1 to W27 does not modify the global fold of Aha1's N-terminal domain spanning T28 to G162 (Figure 3a). Moreover, The CSI data demonstrated that the N-terminal sequence motif of Aha1 spanning M1 to W27 mainly adopts a random coil conformation (Figure 3a). In support of this finding, compared with the dynamics behavior of the core region, extremely high flexibility was observed for the N-terminal sequence motif of Aha1 spanning M1 to W27 (Figure 3b,c).
In line with the finding derived from the AlphaFold2 prediction and the 1H-15N HSQC data, the determind 1H-15N hetNOE values for Aha11-162 at low protein concentration (40 μM) revealed that the N-terminal sequence motif of Aha1 spanning M1 to W27 exhibited an extremely high flexibility. The dynamics feature of Aha1's N-terminal sequence motif spanning M1 to W27 demonstrates that this region is in an unstructured state which independent to the core structure of Aha11-162.
Protein: DP04219 (UniProt O95433)
Region: residues 1	to 27
Annotation: protein folding chaperone (Molecular function)
Evidence: transmission electron microscopy evidence used in manual assertion
Supporting statements:
According to the acquired 1H-15N-HSQC titration data, both the N-terminal fragment M1-W27 and the C-terminal RLF (R336L337F338) motif of Aha1 contributed to the recognition of α-synuclein.
After the confirmation of the interaction between human Aha1 and α-synuclein, ThT assay and transmission electron microscopy (TEM) were applied to test the impact of Aha1 on the aggregation process of α-synuclein. Both the ThT data and the TEM results demonstrated that Aha1 presented an inhibition effect on α-synuclein aggregation (Figure 6d,e).
Aha1 Exhibits Distinctive Dynamics Behavior and Chaperone-Like Activity.
Protein: DP04219 (UniProt O95433)
Region: residues 1	to 16
Annotation: maintenance of unfolded protein (Biological process)
Evidence: fluorescence evidence used in manual assertion
Supporting statements:
During the experiments, the refolding process of MBP without or with the addition of known chaperone SecB, BSA serving as a negative control or Aha1 and its truncations was monitored by the change in the intrinsic tryptophan fluorescence (Figure 5). According to the obtained data, only the full-length Aha1 (Aha11-338) showed a holdase activity against the denatured MBP (Figure 5a–c). The absence of Aha1's N-terminal extension (Aha117-338) fully abolished the holdase activity of the protein, and a similar fluorescence emission profile to that of MBP alone was observed for the MBP:Aha117-338 (molar ration at 1:4) mixture sample (Figure 5c). The fluorescence data indicated that human Aha1 could act as a holdase in vitro, and its N-terminal extension is essential for the holdase activity display.
Human Aha1's N-terminal extension confers it holdase activity in vitro
Protein: DP04219 (UniProt O95433)
Region: residues 1	to 16
Annotation: maintenance of unfolded protein (Biological process)
Evidence: heteronuclear single quantum coherence spectroscopy evidence used in manual assertion
Supporting statements:
As shown in Figure 6a, after an incubation with either SecB or full-length Aha1, the 1H-15N HSQC spectra of MBP showed a feature of unstructured protein with the amide proton resonances crowdedly distributing to the region spanning 7.5–8.5 ppm. Meanwhile, when Aha117-338, Aha121-338, and Aha128-338 were applied, the 1H-15N HSQC data demonstrated that MBP switched from the unfolded state to the folded state.
When Aha11-338W2A was applied, the 1H-15N HSQC spectrum of MBP showed a feature of unstructured protein (Figure 6c).
Protein: DP04220 (UniProt Q9H4H8)
Region: residues 380	to 448
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Recombinant Chica410–478 structure is in agreement with the predicted disorder; its 15N HSQC spectrum (Figure 3a) has a pronounced lack of amide proton chemical shift dispersion, and the CD spectrum shows limited ellipticity at 220 nm and a large negative ellipticity at 200 nm (Figure 3b).
Cross-references: BMRB:26684
Protein: DP04221 (UniProt Q8GJM6)
Region: residues 1	to 18
Annotation: disorder (Structural state)
Evidence: in vitro cleavage assay evidence used in manual assertion
Supporting statements:
Therefore, the first 18 amino acids at the N-terminus were digested within the first minute to produce band A, and further digestion progressed slowly from the C-terminus to produce bands B and C (Figure 1B, C).
Protein: DP04221 (UniProt Q8GJM6)
Region: residues 1	to 18
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
CD spectra of these truncations showed that the N-terminus was indeed disordered on its own, and both the CC domain and CTD maintained α-helical signatures (Figure 2B, Figure 2—figure supplement 1A).
Protein: DP04225 (UniProt A0A7M7G9Q4)
Region: residues 351	to 385
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The UV spectra of the peptides show considerable differences, suggesting a random coil for AmVg(358–392) and a more structured nature for NvVg(351–385) (Fig. 4B,D).
Protein: DP04225 (UniProt A0A7M7G9Q4)
Region: residues 335	to 372
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
NvVg(335–372), with its C-terminus located where the helix occurs in the shorter peptide, shows marked changes, with an attenuation of its NOE pattern (supplementary material Fig. S5 and Table S1). Moreover, the changes are visible in the spectrum as a collapse of the signals (supplementary material Fig. S4C), relative to Fig. 4C. The corresponding CD spectra also indicate a random coil (supplementary material Fig. S4D).
Protein: DP04225 (UniProt A0A7M7G9Q4)
Region: residues 335	to 372
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
NvVg(335–372), with its C-terminus located where the helix occurs in the shorter peptide, shows marked changes, with an attenuation of its NOE pattern (supplementary material Fig. S5 and Table S1). Moreover, the changes are visible in the spectrum as a collapse of the signals (supplementary material Fig. S4C), relative to Fig. 4C.
Cross-references: PDB:2LID
Protein: DP04225 (UniProt A0A7M7G9Q4)
Region: residues 351	to 385
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Solving the structure of the two peptides resulted in an ensemble of outstretched coil structures in the case of AmVg(358–392) (Fig. 6A), whereas that for NvVg(351–385) contains a helix between residues 371 and 377 (Fig. 5A,D, Fig. 6C, Table 1).
Cross-references: PDB:2LID
Protein: DP04226 (UniProt Q93K76)
Region: residues 100	to 109
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The E. coli FocB structure (109 amino acid residues) was determined at a resolution of 1.4 Å by multiwavelength anomalous diffraction [26] from a single crystal of the selenomethionine labeled protein. The crystal comprised two molecules per asymmetric unit. Apart from several residues at the N- and C-terminal ends, all protein residues could be modeled into the electron density. The final model contains residues 10–99 of chain A and residues 10–97 of chain B.
The final R-values, Rwork = 0.196 and Rfree = 0.222, are higher than expected for this resolution. This is probably a result of the additional electron density ascribed to the N- and C-terminal residues of the protein, which were not modeled in the structure because of disorder.
Cross-references: PDB:3M8J
Protein: DP04226 (UniProt Q93K76)
Region: residues 100	to 109
Annotation: flexible C-terminal tail (Disorder function)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The E. coli FocB structure (109 amino acid residues) was determined at a resolution of 1.4 Å by multiwavelength anomalous diffraction [26] from a single crystal of the selenomethionine labeled protein. The crystal comprised two molecules per asymmetric unit. Apart from several residues at the N- and C-terminal ends, all protein residues could be modeled into the electron density. The final model contains residues 10–99 of chain A and residues 10–97 of chain B.
The final R-values, Rwork = 0.196 and Rfree = 0.222, are higher than expected for this resolution. This is probably a result of the additional electron density ascribed to the N- and C-terminal residues of the protein, which were not modeled in the structure because of disorder.
Cross-references: PDB:3M8J
Protein: DP04227 (UniProt P40727)
Region: residues 219	to 229
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
In the case of SalFlhBC the final model comprises residues 229–353 out of 219–383 in the crystallized protein, with a cleavage after Asn269. No electron density was observed for residues 219–228 and 354–383.
Cross-references: PDB:3B0Z
Protein: DP04227 (UniProt P40727)
Region: residues 354	to 383
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
In the case of SalFlhBC the final model comprises residues 229–353 out of 219–383 in the crystallized protein, with a cleavage after Asn269. No electron density was observed for residues 219–228 and 354–383.
Cross-references: PDB:3B0Z
Protein: DP04228 (UniProt O67813)
Region: residues 213	to 231
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The three molecules in the asymmetric unit are very similar, with r.m.s.d.s on pairwise superposition in the range 0.40–0.76 Å. Each molecule consists of two polypeptide chains resulting from proteolytic cleavage after Asn263. In all molecules no electron density was observed for residues 213–231 at the N-terminus; depending on the molecule, two to six residues at the C-terminus were disordered.
Cross-references: PDB:3B1S
Protein: DP04229 (UniProt P9WFG9)
Region: residues 132	to 144
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The 10-stranded β-barrel is well defined in density, except for the solvent-exposed loop 132 to 144, which is presumably disordered in the crystal structure.
Cross-references: PDB:2FWV
Protein: DP04229 (UniProt P9WFG9)
Region: residues 1	to 26
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
This small domain is well conserved in homologous proteins from mycobacteria and corynebacteria (see below) and comprises a long amphipathic α-helix that extends into the solvent and is increasingly disordered toward its N terminus (no density is observed for the first 26 residues in the crystal structure).
Cross-references: PDB:2FWV
Protein: DP04229 (UniProt P9WFG9)
Region: residues 1	to 26
Annotation: flexible N-terminal tail (Disorder function)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
This small domain is well conserved in homologous proteins from mycobacteria and corynebacteria (see below) and comprises a long amphipathic α-helix that extends into the solvent and is increasingly disordered toward its N terminus (no density is observed for the first 26 residues in the crystal structure).
Cross-references: PDB:2FWV
Protein: DP04230 (UniProt P43769)
Region: residues 39	to 58
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
To further improve the spectral appearance, a third clone lacking residues 39-58 was constructed. These residues, which connect the transmembrane helix to the structured domain, were highly flexible based on a lack of 1 H-1 H NOE connectivities and low 15 N R2 relaxation rates (data not shown).
Protein: DP04231 (UniProt P23023)
Region: residues 408	to 427
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Evidence of disorder is provided by four sets of observations. First, resonances in the C-terminal tail exhibit motional narrowing relative to resonances in the UBA folds. Such differences in line widths lead to systematic differences in cross-peak patterns in the three-dimensional HNCACB spectrum (supplemental material). Although, due to line broadening, residues in the UBA fold predominantly exhibit intraresidue cross-peaks with few interresidue Cα and no Cβ cross-peaks, residues in the C-terminal portion of the sex-specific tail (residues 409 – 427) exhibit complete Cα and Cβ connectivities (supplemental material). Second, the pattern of chemical shifts in the C-terminal portion of the tail, unlike those in the UBA fold, is consistent with random coil. Third, the absence of stably folded tail structure is further indicated by an absence of medium and long range NOEs; only sequential and intraresidue NOEs were observed between residues 409 – 427 in three- and four-dimensional 13 C- and 15 N-edited NOESY spectra. Finally, truncation of the tail after residue 412 (i.e. comparison of CTD F -p and CTD F -pΔ) reveals a correspondence of chemical shifts and NOE patterns, demonstrating that the presence or absence of an intact tail does not modulate the folding of the core domain.
The proximal portion of the female-specific tail (residues 398 – 407) contributes to the α-helical structure of UBA fold, whereas the distal portion (residues 408 – 427) is largely disordered.
Cross-references: PDB:2JZ1
Protein: DP04232 (UniProt Q3U0P1)
Region: residues 1	to 10
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Ordered residue range: residues 11 – 39. RPF scores reflect the goodness of fit of the final ensemble of structures (including disordered residues) to the NMR data.
Cross-references: PDB:6E4H
BMRB:27534
Protein: DP04232 (UniProt Q3U0P1)
Region: residues 40	to 60
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Ordered residue range: residues 11 – 39. RPF scores reflect the goodness of fit of the final ensemble of structures (including disordered residues) to the NMR data.
Cross-references: PDB:6E4H
BMRB:27534
Protein: DP04233 (UniProt P9WMI9)
Region: residues 106	to 118
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The NH cross-peaks of the C-terminal region (residues 106 –118) are all clustered in the typical spectral region of unstructured proteins and are
highly flexible, displaying backbone mobility in the nanosecond to picosecond time scale, considerably faster than the overall protein tumbling rate (supplemental Fig. S4). Consequently this region is characterized by a very low number of long range NOEs (supplemental Fig. S5).
Cross-references: PDB:2JSC
Protein: DP04233 (UniProt P9WMI9)
Region: residues 106	to 118
Annotation: flexible C-terminal tail (Disorder function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The NH cross-peaks of the C-terminal region (residues 106 –118) are all clustered in the typical spectral region of unstructured proteins and are highly flexible, displaying backbone mobility in the nanosecond to picosecond time scale, considerably faster than the overall protein tumbling rate (supplemental Fig. S4). Consequently this region is characterized by a very low number of long range NOEs (supplemental Fig. S5).
Cross-references: PDB:2JSC
Protein: DP04234 (UniProt O88574)
Region: residues 130	to 142
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The vast majority of the residues of both mSin3A PAH3 and SAP30 SID adopt well defined conformations, although the four N-terminal and two C-terminal residues of mSin3A PAH3 as well as the 13 N-terminal and 11 C-terminal residues of SAP30 SID are disordered in the complex.
Cross-references: PDB:2LD7
BMRB:17653
Protein: DP04234 (UniProt O88574)
Region: residues 210	to 220
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The vast majority of the residues of both mSin3A PAH3 and SAP30 SID adopt well defined conformations, although the four N-terminal and two C-terminal residues of mSin3A PAH3 as well as the 13 N-terminal and 11 C-terminal residues of SAP30 SID are disordered in the complex.
Cross-references: PDB:2LD7
BMRB:17653
Protein: DP04235 (UniProt Q03390)
Region: residues 128	to 186
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
To confirm this observation, we preformed CD spectroscopy on free Vps60(128–186), where negative absorption at ∼200 nm shows a random coil conformation (Fig. 2E).
Protein: DP04235 (UniProt Q03390)
Region: residues 128	to 186
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The two-dimensional NMR 1H-15N HSQC experiment with Vps60(128–186) in its free state (Fig. 2D) suggests that Vps60(128–186) in its free state is disordered because the cross-peaks are not dispersed, localizing mainly in the region between 8.0 and 8.5 ppm.
Protein: DP04235 (UniProt Q03390)
Region: residues 128	to 186
Annotation: disorder to order (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Upon binding to Vta1NTD, the cross-peaks in two-dimensional NMR 1H-15N HSQC of Vps60(128–186) became dispersed (Fig. 2D), suggesting that Vps60(128–186) folds into an ordered structure, coinciding with the structure determined below. This demonstrates that Vta1NTD binding stabilizes Vps60 helix conformation.
Cross-references: PDB:2LUH
Protein: DP04235 (UniProt Q03390)
Region: residues 140	to 183
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The side chains of the residues located at Vps60(128–186) helix α4′, including Met-140′, Leu-141′, Leu-143′, Ile-144′, Leu-150′, Val-153′, and Leu-154′, are inserted into the groove of Vta1NTD helices 5 and 7. The Vps60(128–186) Ile-144′ side chain has a hydrophobic interaction with the Vta1NTD Ile-91, Leu-94, and Met-98 side chains, as Pro-171 functions in the complex Vps4A-CHMP6(168–179) (21). Besides the hydrophobic interactions, complementary salt bridges are also formed by two of the adjacent conserved Vps60(128–186) residues (Glu-149′ and Glu-152′) (Fig. 3B).
Moreover, the side chains of Vta1NTD Asn-95 and Asn-102 may form hydrogen bond interactions with the main chains of Met-140′ and Gln-146′, respectively. The second site on Vta1NTD binds mainly to the linker between α4′ and α5′ of Vps60(128–186) (Fig. 3C), most prominently to the side chains of Leu-163′ and Ile-166′. This site is also mainly hydrophobic and is lined by Tyr-153, Ile-156, and Tyr-157 and the aliphatic chains of Lys-149, Lys-152, and Lys-160, except that the side chain of Glu-162′ forms a complementary salt bridge with Lys-149.
The third binding site on Vta1 contacts Vps60(128–186) helix α5′ (Fig. 3D), including previously identified Vta1 residues important for Vps60 interaction, Trp-122 and Lys-152 (27, 29).
Cross-references: PDB:2LUH