LiteFold/DisProt huggingface dataset:
Protein: DP04129 (UniProt P42858)
Region: residues 1	to 90
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The 25Q form of Httex1 lacks persistent protection from exchange by NMR at pH 7.5, suggesting an absence of persistent hydrogen bonds (Fig. S2).
Fig. 2. Httex1 lacks persistent hydrogen bonding with 25Q or 46Q. (a) 1H,15N HSQC of Httex1 25Q protein fragment in H2O versus D2O-based sodium acetate buffer (150 mM; pH 4).
 The first four residues within Httex1 have negative NOE values, which is consistent with high mobility of the N-terminus (Fig. 3c). The next 13 acids N-terminal to the polyQ region as well as the arc of glutamine backbone resonances displayed positive NOE values mostly between 0.4 and 0.6. The C-terminal proline-rich region shows 15N{1H}-NOE values that are < 0.4. These observations suggest that, while the N17 and polyQ tract are not ordered in the canonical sense, there is a persistent preference for conformational rigidity implying low-amplitude conformational fluctuations, especially within the N17 and polyQ regions.
Analysis of the smoothed ΔCα–ΔCβ values [40] indicated localized regions of distinct secondary structure (values ≥ 1) and regions lacking stable structure but with a tendency toward some α-helical structure throughout the N-terminal and polyQ regions of Httex1 (Fig. 3b) [39], [40], [41]. This result is consistent with findings that portions of the polyQ region are able to form α-helices [20], [42], [43].
The C-terminal sequence displayed clear preferences for extended structures (Fig. 3b). Overall, these results suggest that Httex1 adopts a heterogeneous ensemble of conformations with localized preferences for specific secondary structure motifs.
Cross-references: BMRB:27161
Protein: DP04129 (UniProt P42858)
Region: residues 1	to 90
Annotation: disorder (Structural state)
Evidence: protein hydrogen-deuterium exchange mass spectrometry evidence used in manual assertion
Supporting statements:
Fig. 2. Httex1 lacks persistent hydrogen bonding with 25Q or 46Q. (a) 1H,15N HSQC of Httex1 25Q protein fragment in H2O versus D2O-based sodium acetate buffer (150 mM; pH 4). Labeled residues are observed after HDX. (b) HDX of Httex1 25Q and 46Q were measured over 10 min using MS at both pH 7.5 and pH 4. Exchange plateaued at ~ 80% (due to back-exchange in the protonated solvent during chromatography separation prior to MS).
Given that the HDX-MS results for both 25Q and 46Q forms imply a lack of stable hydrogen bonding, we focused on the 25Q variant for the remainder of our biophysical assays to probe additional structural details within Httex1. In an effort to rule out weak hydrogen bonding, we also investigated HDX combined with NMR of Httex1 25Q at pH 4. Under these conditions, the exchange of a freely exposed peptide group is approximately 3 orders of magnitude slower than at pH 7.5 [26]. For Httex1 25Q, seven backbone amide hydrogens were found to be protected from deuteration at pH 4.
Our HDX data are consistent with a lack of persistent hydrogen bonding. This lack of hydrogen bonding may result from conformational heterogeneity within the polyQ domain and throughout Httex1, as predicted by the simulations, or from fully solvated, rod-like geometries with a preference for polyproline II conformations.
By measuring HDX-MS, we find that there is no clear evidence for changes in solvent protection, to imply the presence of stable beta-sheeted hydrogen bonds, with increased polyQ length.
Protein: DP04129 (UniProt P42858)
Region: residues 1	to 87
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The difference spectra for both proteins exhibit characteristic minima at 208 and 222 nm indicating increased formation of alpha-helical structure at lower temperatures, which is more pronounced for Q46 HTT protein (Fig. 6A), and all spectra (Fig. 5B and C) show an isosbestic point near 203 nm, which is commonly observed for conversions between random coil and alpha-helical structure.
The consistent isosbestic point across temperatures (Fig. 5B and C and 6B and C) together with the typically alpha-helical difference spectra (Fig. 6A and D) suggest that the conformational changes are dominated by a helix-coil transition.
Our CD studies indicate that HTT exon 1 can take up an extensive helical structure that likely includes the N17 as well as substantial portions of the polyQ region. This notion is in agreement with prior crystallographic studies, which showed that alpha-helical structure could extend from the N17 into the polyQ region in some, but not all structures [24] [25 [24]]. Our data indicate that the observed alpha-helical and unfolded structures are in equilibrium with one another.
Protein: DP04129 (UniProt P42858)
Region: residues 1	to 87
Annotation: disorder (Structural state)
Evidence: analytical ultracentrifugation evidence used in manual assertion
Supporting statements:
In contrast to TRX-tag alone, the exon 1 fusion proteins with normal or expanded poly(Gln) are nonglobular by several measures, including anomalous migration in size exclusion chromatography and high frictional ratios (f/f0) in sedimentation velocity ultracentrifugation (Table 1).
Protein: DP04129 (UniProt P42858)
Region: residues 1	to 87
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The CD spectra of HD exon 1 with different lengths of poly(Gln) (Fig. 2a) resemble the spectra of disordered proteins. In particular, the spectra are reminiscent of denatured collagen (21), which has a similar high imino acid (e.g., proline) content.
These results are consistent with a random-coil structure for both normal and expanded poly(Gln) tracts within HD exon 1. Moreover, the data suggest that flanking sequences in HD exon 1 (17 residues at the N terminus and 49 residues at the C terminus) do not adopt stable α or β secondary structures and that their presence does not induce a global transition in the poly(Gln) tract above the pathologic threshold.
Protein: DP04129 (UniProt P42858)
Region: residues 1	to 87
Annotation: disorder (Structural state)
Evidence: gel-filtration evidence used in manual assertion
Supporting statements:
During purification of huntingtin exon 1 fusion proteins, we observed anomalous migration by gel filtration chromatography such that huntingtin exon 1 fusion proteins appeared to migrate as higher molecular weight proteins (e.g., dimers) when compared with molecular weight standards of globular proteins.
Protein: DP04129 (UniProt P42858)
Region: residues 1	to 87
Annotation: disorder (Structural state)
Evidence: small-angle X-ray scattering evidence used in manual assertion
Supporting statements:
By contrast, the Kratky plots for HD-16Q and HD-39Q were broader, with less degrease at higher scattering angles, consistent with flexible or unfolded proteins [21, 22] (Fig. 5b).
(b) HD-16Q and HD-39Q showed a plateau at higher q values, suggesting that these proteins include disordered regions, with decreasing globular character as the polyQ repeat length increased.
Thus a combination of equilibrium gel filtration chromatography, DLS, and SAXS data are consistent with a linear lattice mode of recognition of unstructured polyQ for both 3B5H10 and MW1 antibodies.
Protein: DP04129 (UniProt P42858)
Region: residues 1	to 87
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Next, we used the MREHttex1 values to estimate the number of nominal residues that convert from a random coil into an α-helical conformation when transitioning from 37 to −10 °C. These estimates were gradually increasing with Q-length and ranged from 11 amino acids for Q7 to 32 amino acids for Q55 (Fig. 1f). Inasmuch as the number of residues becoming α-helical was often larger than 17 (for Q25 and higher), regions outside of the N17 must have contributed to the observed helicity.
Still most amino acids in the construct remain to be classified as random coil.
Protein: DP04129 (UniProt P42858)
Region: residues 1	to 87
Annotation: disorder (Structural state)
Evidence: site-directed spin-labelling electron paramagnetic resonance evidence used in manual assertion
Supporting statements:
The spectra for 11R1 (N17) and 35R1 (polyQ) shared the interesting similarity that they had multiple components that became more clearly resolved at lower temperatures. One of these spectral components indicated a highly mobile structural state (Fig. 2b, denoted m), whereas another one is characteristic of a more immobilized and stable structural state (denoted i). In contrast, 81R1 lacked the immobilized spectral component and was dominated solely by the mobile spectral component.
The lack of immobilization for these sites indicated relatively dynamic regions that do not engage in significant tertiary packing interactions. The collective occurrence of pronounced multicomponent EPR spectra that were exclusively found at all N-terminal sites up to residue 35 strongly suggested that this region was characterized by at least two co-existing structural populations. One of these populations was dynamic and largely disordered, and the other was more ordered, likely corresponding to the α-helical structure observed by CD.
When comparing amplitudes obtained at the same temperature, the labeling sites in the N17 (Fig. 2c, blue) tended to have the lowest amplitudes, indicative of lowest mobility/highest order in this region. In contrast, the largest amplitudes (highest mobilities) were found in the PRD.
EPR spin-labeling analysis of Trx–Httex1(Q46) reveals the co-existence of two structural states in the N17 and the adjacent N-terminal portion of polyQ. One of these states is highly dynamic and largely disordered, whereas the other state is ordered, consistent with α-helical structure.
Protein: DP04129 (UniProt P42858)
Region: residues 1	to 87
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
If all polyQ residues of Trx–Httex1(Q46) were structurally homogeneous, a single intense resonance would dominate the NMR spectra. In Trx–Httex1(Q46), dominating backbone HN and side-chain H2N resonances were indeed detected (Fig. S3). However, the strongest HN backbone resonance clearly was not 46 times stronger than signals from other residues, suggesting that the polyQ region was not structurally homogeneous.
As a simple measure of relative backbone dynamics along the Trx–Httex1(Q7) sequence, we compared signal intensities (Fig. 5a). The linker resonances exhibited the highest intensities, verifying the efficient uncoupling of the Trx domain from Httex1(Q7) already indicated in the aforementioned EPR (Fig. S2) and NMR (Fig. 3) experiments. The PRD also exhibited high albeit variable dynamics in overall accordance with EPR analysis (Fig. 2).
NMR spectroscopy further reveals that polyQ partakes in this temperature-dependent α-helix formation. However, helical structure is not uniformly adopted throughout the polyQ region. Rather, multiple NMR resonances are observed for the polyQ region, each arising from sequence populations of different helical content.
Cross-references: BMRB:27636
Protein: DP04129 (UniProt P42858)
Region: residues 1	to 88
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The secondary chemical shift (SCS) analysis using a neighbor-corrected random coil database (Nielsen and Mulder, 2018) indicates that both N17 and the poly-Q tract are enriched in α-helical conformations, although this propensity is not homogeneous (Figure 2D). Helicity increases along N17, reaching its maximum at the first glutamine, Q18, and subsequently decreases smoothly. A transition is observed at Q29, which adopts a small and negative SCS value. This extends to the following three glutamines, indicating the presence of random coil or slightly extended conformations.
Three of these ensembles present α helices that encompass the last residues of N17 and the first residues of the poly-Q. No persistent turns in the residues connecting both domains are observed, which would otherwise yield a strong signature in the CS profile. As a consequence, H16 should be considered as an elongated flexible particle, in contrast to the previously proposed compact tadpole-like model (Newcombe et al., 2018, Warner et al., 2017).
This observation demonstrates that the poly-P tract in httex1 exerts a strong conformational perturbation on the neighboring glutamines by enriching the ensemble with extended conformations, which break the inherent helical propensity of the poly-Q.
Protein: DP04129 (UniProt P42858)
Region: residues 1	to 88
Annotation: disorder (Structural state)
Evidence: small-angle X-ray scattering evidence used in manual assertion
Supporting statements:
To further confirm that the overall extendedness is inherent to httex1, we performed an equivalent SAXS analysis for H16 (FigS. 3a, S3 and Table S2). Not surprisingly, the resulting SAXS profile of H16 indicated that the protein is a smaller particle (Rg=32.9 ± 0.2 Å and Dmax = 126 Å) in solution than H46 (Table S2). Importantly, H16 retained SAXS features corresponding to an extended and flexible particle observed for H46.
Protein: DP04129 (UniProt P42858)
Region: residues 1	to 88
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Similar to H16 (ref. 23), which also contained a fused sfGFP, the spectrum revealed that, while peaks from N17 and the PRR were well dispersed, glutamine frequencies remained poorly resolved (Fig. 1b).
After modeling the sfGFP and the C-terminal His-tag to the individual conformations, the NMR-optimized ensemble was further refined by integrating the SAXS data with the ensemble optimization method (EOM)30,31. Subensembles selected with EOM yielded an excellent fit to the experimental profile (χ2 = 0.2) (Fig. 3a). The resulting radius of gyration (Rg) distribution was broad, indicating that H46 is a highly flexible particle in solution and slightly more extended than the CS-derived ensemble (Extended Data Fig. 3).
The refined ensemble indicates that H46 is a flexible elongated particle in solution and that the overall size is correlated with the length of the poly-Q (Fig. 3c). Our structural model is in contradiction to previously reported compact models of httex1 stabilized by extensive fuzzy contacts between N17 and poly-Q14,15,46,47. Despite the overall extendedness of httex1, our description requires a large degree of disorder, explaining the fluorescence transfer efficiency observed in smFRET14 and the lack of permanent hydrogen bonds in NMR hydrogen deuterium exchange experiments, which have been argued to support the compact model15.
Protein: DP04129 (UniProt P42858)
Region: residues 1	to 87
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
This assignment allowed us to also measure site-specific 15N R1 and R2 relaxation rates as well as residual 1H-15N dipolar couplings that confirmed the highly dynamic nature of these residues in the context of the fibril (15).
Overall, the PRD stays relatively extended throughout the simulations with two stable PPII helices, P10 and P11, a more variable L17 linker region, and a very flexible C-terminal C12 region. Nevertheless, the L17 and, to a lesser extent, the C12 region have average dihedral angles compatible with a PPII helical or β-sheet conformation and are extended for most of the simulation, indicating that these regions are rather imperfect PPII helices than completely disordered. This extended PRD is also compatible with our previous observation that unbundled huntingtin (HTT) fibrils are spaced consistent with fibrils being held apart by extended polyproline bristles (12).
Our finding that the PRD is mostly extended is compatible with the tadpole model of the HTTex1 monomer by Newcombe and co-workers in which the N17 and polyQ domains are more compact, and the PRD forms the extended tail of a tadpole-like structure (24).
Protein: DP04129 (UniProt P42858)
Region: residues 1718	to 1732
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
To gain further insight into the structure of Q23-HTT in the apo state, we generated and fitted atomic models to our cryo-EM map of Q23-HTT utilizing the structure of HTTHAP40 in complex with HAP40 via an integrative modeling strategy. The HTTHAP40 model was split into three rigid domains, N-HEAT, Bridge, and C-HEAT, based on the fact that these three domains are weakly connected by unresolved flexible regions (residues 1,713–1,729 separating N-HEAT and Bridge domains, and residues 2,062–2,092 separating Bridge and C-HEAT domains) in HTT HAP40 structure.
UniProt numbering is slightly different from the authors' numbering.
Cross-references: PDB:6RMH
EMDB:EMD-4937
Protein: DP04129 (UniProt P42858)
Region: residues 1718	to 1732
Annotation: flexible linker (Disorder function)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
To gain further insight into the structure of Q23-HTT in the apo state, we generated and fitted atomic models to our cryo-EM map of Q23-HTT utilizing the structure of HTTHAP40 in complex with HAP40 via an integrative modeling strategy. The HTTHAP40 model was split into three rigid domains, N-HEAT, Bridge, and C-HEAT, based on the fact that these three domains are weakly connected by unresolved flexible regions (residues 1,713–1,729 separating N-HEAT and Bridge domains, and residues 2,062–2,092 separating Bridge and C-HEAT domains) in HTT HAP40 structure.
UniProt numbering is slightly different from the authors' numbering.
Cross-references: PDB:6RMH
EMDB:EMD-4937
Protein: DP04129 (UniProt P42858)
Region: residues 2067	to 2095
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
To gain further insight into the structure of Q23-HTT in the apo state, we generated and fitted atomic models to our cryo-EM map of Q23-HTT utilizing the structure of HTTHAP40 in complex with HAP40 via an integrative modeling strategy. The HTTHAP40 model was split into three rigid domains, N-HEAT, Bridge, and C-HEAT, based on the fact that these three domains are weakly connected by unresolved flexible regions (residues 1,713–1,729 separating N-HEAT and Bridge domains, and residues 2,062–2,092 separating Bridge and C-HEAT domains) in HTT HAP40 structure.
UniProt numbering is slightly different from the authors' numbering.
Cross-references: PDB:6RMH
EMDB:EMD-4937
Protein: DP04129 (UniProt P42858)
Region: residues 2067	to 2095
Annotation: flexible linker (Disorder function)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
To gain further insight into the structure of Q23-HTT in the apo state, we generated and fitted atomic models to our cryo-EM map of Q23-HTT utilizing the structure of HTTHAP40 in complex with HAP40 via an integrative modeling strategy. The HTTHAP40 model was split into three rigid domains, N-HEAT, Bridge, and C-HEAT, based on the fact that these three domains are weakly connected by unresolved flexible regions (residues 1,713–1,729 separating N-HEAT and Bridge domains, and residues 2,062–2,092 separating Bridge and C-HEAT domains) in HTT HAP40 structure.
UniProt numbering is slightly different from the authors' numbering.
Cross-references: PDB:6RMH
EMDB:EMD-4937
Protein: DP04129 (UniProt P42858)
Region: residues 2067	to 2090
Annotation: disorder (Structural state)
Evidence: protein hydrogen-deuterium exchange mass spectrometry evidence used in manual assertion
Supporting statements:
Another region showing high exchange rate is around aa 2,000–2,200 and is located at the interface between the Bridge and C-HEAT domains, which is consistent with our observation that the Bridge domain serves as pivot joints making HTT conformationally flexible.
Figure S5. Related to Figure 5. HDX-MS Experiments. (A) The HDX-MS deuteration map of Q23- (A), and Q78-HTT (B). Total 147 peptides detected and the coverage was 42.7%. The N-HEAT, bridge, and the C-HEAT domain were indicated by blue, orange, and red boxes, respectively.
The authors refer to a 200 residues long region of high exchange rate, but for the stringency of the annotation only a shorter region with higher than 60% exchange rate after 45 min was curated. This greatly overlaps with the missing electron densities in the cryo-EM map (c.f. DP04129r024), which validates the interpretation.
Protein: DP04129 (UniProt P42858)
Region: residues 2067	to 2090
Annotation: flexible linker (Disorder function)
Evidence: protein hydrogen-deuterium exchange mass spectrometry evidence used in manual assertion
Supporting statements:
Another region showing high exchange rate is around aa 2,000–2,200 and is located at the interface between the Bridge and C-HEAT domains, which is consistent with our observation that the Bridge domain serves as pivot joints making HTT conformationally flexible.
Figure S5. Related to Figure 5. HDX-MS Experiments. (A) The HDX-MS deuteration map of Q23- (A), and Q78-HTT (B). Total 147 peptides detected and the coverage was 42.7%. The N-HEAT, bridge, and the C-HEAT domain were indicated by blue, orange, and red boxes, respectively.
The authors refer to a 200 residues long region of high exchange rate, but for the stringency of the annotation only a shorter region with higher than 60% exchange rate after 45 min was curated. This greatly overlaps with the missing electron densities in the cryo-EM map (c.f. DP04129r024), which validates the interpretation.
Protein: DP04129 (UniProt P42858)
Region: residues 407	to 664
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Besides the polyQ tract, a large portion of N-HEAT domain (aa 409–666 in Q23-HTT) in the high-resolution cryo-EM structure is unstructured in addition to several small regions in other domains.
UniProt numbering is slightly different from the authors' numbering.
Cross-references: EMDB:EMD-4937
PDB:6RMH
Protein: DP04129 (UniProt P42858)
Region: residues 419	to 432
Annotation: phosphorylation display site (Disorder function)
Evidence: liquid chromatography coupled with tandem mass spectrometry evidence used in manual assertion
Supporting statements:
Besides the polyQ tract, a large portion of N-HEAT domain (aa 409–666 in Q23-HTT) in the high-resolution cryo-EM structure is unstructured in addition to several small regions in other domains.
UniProt numbering is slightly different from the authors' numbering.
Interestingly, many residues in the large unstructured region in N-HEAT domain are shown to be phosphorylated, and these modifications seem to modify HTT function.
Several HTT phosphorylation sites were identified by MS and are listed along the length of the HTT amino acid sequence.
Phosphosites on Figure 6A in the IDR streching between aa. 407-664 within the N-HEAT domain: pS421 and pS434. With UniProt numbering these correspond to pS419 and pS432.