LiteFold/DisProt huggingface dataset:
Protein: DP04161 (UniProt B7UM99)
Region: residues 509	to 515
Annotation: SH2 domain binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Using the C-terminal SH2 domain (C-SH2) of phosphatase SHP-1, we identified, by NMR, that residues surrounding the unphosphorylated Y511 ITIM-like motif are involved in binding SH2 domains (Fig. 6a). Binding to C-Tir caused a selective loss of the backbone amide cross-peaks intensities, mainly from residues A512LLA515 (Fig. 6b). Visible NMR signals retained low dispersion, indicating that the protein remains mostly disordered and flexible in the complex
The Y511 points to the canonically defined “pTyr pocket”, and the SH2 recognizes the hydrophobic Leu residue in position +3 via the “specific pocket”70. Moreover, residue in position −2 interacts with αA, forming a “three-pronged plug” interaction altogether.
Protein: DP04161 (UniProt B7UM99)
Region: residues 454	to 511
Annotation: SH2 domain binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
With the resonance re-assignment of pC-Tir, we identified that upon phosphorylation, all tyrosine sites interact with C-SH2, and not exclusively the Y511-based motif as observed for unphosphorylated C-Tir.
Phosphorylation enhanced the binding to Y511 site by ~10 fold and enabled other tyrosine-based motifs to engage C-SH2. Among the four pYs, the resonances around pY454 were less broadened during the titration, suggesting lower local binding. The remaining pYs bind C-SH2 with similar strength (~3-9μM). Overall, phosphorylation at multiple tyrosine sites provides various SH2 docking sites and reinforces the role of Tir as a scaffolding hub.
Protein: DP04161 (UniProt B7UM99)
Region: residues 483	to 515
Annotation: lipid binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
In the presence of bicelles containing DMPC lipids, we observed a marked residue-specific decrease in the ratio of NMR signals around Y511, yet more subtle, also around Y483 (Fig. 8). This finding shows that C-Tir does interact with non-charged lipid bilayers predominantly via its Y511-based motif, including mostly hydrophobic residues, such as A512LLA515.
Protein: DP04161 (UniProt B7UM99)
Region: residues 35	to 77
Annotation: protein binding (Molecular function)
Evidence: co-purification evidence used in manual assertion
Supporting statements:
Tir fragments containing residues 23–80, 32–80, and 35–77 co-purified CesT as seen by Ni2+-affinity pull-down and immunoblotting (Fig 1B and S1A Fig), whereas CesT alone was never pulled-down in the absence of Tir by the Ni2+-affinity resin, thus confirming specificity of our assay. When these Tir fragments were truncated further to residues 32–73, 37–80, and 37–73, they lost the ability to co-purify with CesT (Fig 1B).
Protein: DP04161 (UniProt B7UM99)
Region: residues 35	to 53
Annotation: chaperone binding (Molecular function)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
Tir32-80 adopts minimal regular secondary structure that is limited to two small β-strands, β1’ and β2’ (Fig 2A). The Tir32-80 fragment binds CesT138 in two distinct locations and is separated by a break in the peptide chain likely due to residue mobility in the crystal. Tir residues 35–53 adopt a β-hairpin-like fold and extend the 5-stranded β-sheet core of CesT138, while also being pinched between α1 and orthogonally below by α3 of CesT138 (Fig 2A).
The β-motif was originally identified in the SipA-InvB complex [31, 32], but appears to be a conserved mode of binding present in all class I chaperone-effector complexes [33].
Cross-references: PDB:5WEZ
Protein: DP04161 (UniProt B7UM99)
Region: residues 35	to 53
Annotation: order (Structural state)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
Tir32-80 adopts minimal regular secondary structure that is limited to two small β-strands, β1’ and β2’ (Fig 2A). The Tir32-80 fragment binds CesT138 in two distinct locations and is separated by a break in the peptide chain likely due to residue mobility in the crystal. Tir residues 35–53 adopt a β-hairpin-like fold and extend the 5-stranded β-sheet core of CesT138, while also being pinched between α1 and orthogonally below by α3 of CesT138 (Fig 2A).
The β-motif was originally identified in the SipA-InvB complex [31, 32], but appears to be a conserved mode of binding present in all class I chaperone-effector complexes [33].
Cross-references: PDB:5WEZ
Protein: DP04161 (UniProt B7UM99)
Region: residues 35	to 53
Annotation: disorder to order (Structural transition)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
Tir32-80 adopts minimal regular secondary structure that is limited to two small β-strands, β1’ and β2’ (Fig 2A). The Tir32-80 fragment binds CesT138 in two distinct locations and is separated by a break in the peptide chain likely due to residue mobility in the crystal. Tir residues 35–53 adopt a β-hairpin-like fold and extend the 5-stranded β-sheet core of CesT138, while also being pinched between α1 and orthogonally below by α3 of CesT138 (Fig 2A).
The β-motif was originally identified in the SipA-InvB complex [31, 32], but appears to be a conserved mode of binding present in all class I chaperone-effector complexes [33].
Cross-references: PDB:5WEZ
Protein: DP04161 (UniProt B7UM99)
Region: residues 65	to 75
Annotation: chaperone binding (Molecular function)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
Tir residues 65–75 are bound along the concave surface of the β-sheet core of CesT138 (Fig 2A).
Specifically, Tir residues I38 (purple), L44 (cyan), and L49 (cyan) anchor the β-hairpin-like peptide to CesT138 (Fig 2D); and L69 plus three additional proline residues make a second point of contact with the β-sheet core of CesT138 (Fig 2E).
Cross-references: PDB:5WEZ
Protein: DP04161 (UniProt B7UM99)
Region: residues 490	to 550
Annotation: chaperone binding (Molecular function)
Evidence: bacterial 2-hybrid assay evidence used in manual assertion
Supporting statements:
T18 fused to Tir490-550 showed a strong interaction with CesT fused to T25 by BACTH assays (Fig 4B), and was able to pull-down CesT in co-expression pull-down experiments (Fig 4D), confirming this site as a second CesT-binding region.
Furthermore, both of the Tir CesT-binding regions have a conserved sequence motif distinct from the known chaperone binding β-motif.
Protein: DP04161 (UniProt B7UM99)
Region: residues 490	to 550
Annotation: chaperone binding (Molecular function)
Evidence: co-purification evidence used in manual assertion
Supporting statements:
T18 fused to Tir490-550 showed a strong interaction with CesT fused to T25 by BACTH assays (Fig 4B), and was able to pull-down CesT in co-expression pull-down experiments (Fig 4D), confirming this site as a second CesT-binding region.
Furthermore, both of the Tir CesT-binding regions have a conserved sequence motif distinct from the known chaperone binding β-motif.
Protein: DP04163 (UniProt P32120-2)
Region: residues 350	to 393
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The PDB structure shows this region lacks electron density, indicating it is disordered.
Cross-references: PDB:5TV1
Protein: DP04163 (UniProt P32120-2)
Region: residues 349	to 385
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The carboxy terminus folds back toward the N-terminal domain, becomes unstructured for ~35 residues and forms a highly conserved tripartite interaction with the N-terminal domain consisting of two hydrophobic interactions with β-strand I and α-helix I and one buried ion pair constituting part of the main arrestin phosphate sensor, the polar core20; 28.
Cross-references: PDB:3P2D
Protein: DP04164 (UniProt O94885)
Region: residues 396	to 564
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The 1H-15N HSQC of the SPIDER showed a narrow dispersion of peaks characteristic of a disordered protein (Fig. 3), confirming the prediction.
We designed a construct that comprises residues 396–564 in SASH1.
Cross-references: BMRB:51747
Protein: DP04165 (UniProt E7CC86)
Region: residues 147	to 166
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The deviations of the Cα and Cβ chemical shifts (Δδ) from the mean random-coil values were calculated and the difference ΔδCα – ΔδCβ was plotted against the OnubPBP3 sequence to obtain the secondary structural information in the protein (Fig. 3). This plot shows that OnubPBP3 contained seven α-helices starting with the N-terminus as the first helix. The C-terminus of the protein was predicted to be unstructured.
Based on TALOS + calculations, the secondary structural elements in OnubPBP3 consist of six helices: 3–22, 27–34, 46–58, 71–79, 84–96, 108–124. The C-terminus of OnubPBP3 is unstructured, in stark contrast to the well-formed helix of the C-terminus of OfurPBP2.
The measured construct is the uniprot protein without the N-terminal 22 aa long signal peptide. The last helical amino acid mentioned (position 124) is therefore equivalent to UniProt position 146 and the disordered tail starts from the next residue (147-166).
Cross-references: BMRB:51937
Protein: DP04165 (UniProt E7CC86)
Region: residues 147	to 166
Annotation: flexible C-terminal tail (Disorder function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The deviations of the Cα and Cβ chemical shifts (Δδ) from the mean random-coil values were calculated and the difference ΔδCα – ΔδCβ was plotted against the OnubPBP3 sequence to obtain the secondary structural information in the protein (Fig. 3). This plot shows that OnubPBP3 contained seven α-helices starting with the N-terminus as the first helix. The C-terminus of the protein was predicted to be unstructured.
Based on TALOS + calculations, the secondary structural elements in OnubPBP3 consist of six helices: 3–22, 27–34, 46–58, 71–79, 84–96, 108–124. The C-terminus of OnubPBP3 is unstructured, in stark contrast to the well-formed helix of the C-terminus of OfurPBP2.
The measured construct is the uniprot protein without the N-terminal 22 aa long signal peptide. The last helical amino acid mentioned (position 124) is therefore equivalent to UniProt position 146 and the disordered tail starts from the next residue (147-166).
Cross-references: BMRB:51937
Protein: DP04166 (UniProt P56536)
Region: residues 325	to 338
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Information on the secondary structure of the kinesin neck fragment based on secondary chemical shifts and NOEs is summarized in Fig. 2A. The beginning of the neck linker is disordered but residues T330-L335 show β-strand propensity. The neck coiled coil runs between residues A339 and R371 (Fig. 2A) and shows a typical HN chemical shift periodicity (Kaplan et al. 2017), except for the connection between the first and second heptad (Fig. 2B) that has polar residues at the a and d heptad positions (Kozielski et al. 1997). By contrast, the linker region preceding the coiled coil has a two-residue periodicity suggestive of a β-strand conformation.
Figure 2D shows chemical shift-based S2 order parameters for the neck domain calculated with Talos-N (Shen and Bax 2015). Note the β-strand region in the linker forms an island of intermediate S2 values of ~ 0.7, suggesting that this segment is not completely disordered but separated from the coiled coil by a dynamic hinge.
The neck linker is disordered relative to the coiled coil (Figs. 2D and 3A) with no NOEs observed between the two segments. Nevertheless, the linker has a 3–6 residue segment of relatively well defined extended β-strand structure, lacking a distinct orientation relative to the coiled coil due to flexibility of the E336-A339 hinge connecting the two segments.
Cross-references: BMRB:52075
PDB:8TT7
Protein: DP04166 (UniProt P56536)
Region: residues 325	to 338
Annotation: flexible linker (Disorder function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Information on the secondary structure of the kinesin neck fragment based on secondary chemical shifts and NOEs is summarized in Fig. 2A. The beginning of the neck linker is disordered but residues T330-L335 show β-strand propensity. The neck coiled coil runs between residues A339 and R371 (Fig. 2A) and shows a typical HN chemical shift periodicity (Kaplan et al. 2017), except for the connection between the first and second heptad (Fig. 2B) that has polar residues at the a and d heptad positions (Kozielski et al. 1997). By contrast, the linker region preceding the coiled coil has a two-residue periodicity suggestive of a β-strand conformation.
Figure 2D shows chemical shift-based S2 order parameters for the neck domain calculated with Talos-N (Shen and Bax 2015). Note the β-strand region in the linker forms an island of intermediate S2 values of ~ 0.7, suggesting that this segment is not completely disordered but separated from the coiled coil by a dynamic hinge.
The neck linker is disordered relative to the coiled coil (Figs. 2D and 3A) with no NOEs observed between the two segments. Nevertheless, the linker has a 3–6 residue segment of relatively well defined extended β-strand structure, lacking a distinct orientation relative to the coiled coil due to flexibility of the E336-A339 hinge connecting the two segments.
This disordered linker connects the head domain of dimeric kinesin (missing in this construct) and the neck coiled-coil region.
Cross-references: BMRB:52075
PDB:8TT7
Protein: DP04166 (UniProt P56536)
Region: residues 325	to 338
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy-based hydrogen-deuterium exchange evidence used in manual assertion
Supporting statements:
Figure 2 C summarizes hydrogen exchange (HX) data for the neck domain measured for a sample at pH* 6.1 and a temperature of 23 °C, using 1H-15N HSQC spectra recorded as a function of incubation time in D2O.
Figure 2C clearly shows that the neck linker is not protected from HD exchange.
Cross-references: BMRB:52075
Protein: DP04167 (UniProt Q8N6T7)
Region: residues 284	to 355
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
A biochemical study suggests that the intrinsically disordered C-terminal domain of SIRT6 is necessary for establishing tight interaction of SIRT6 with the nucleosome (Liu et al., 2020) via its ability to bind DNA.
The cryo EM structure of the Nucleosome Bound SIRT6 shows this region lacks electron density, indicating it is disordered.
Cross-references: PDB:8OF4
EMDB:16845
Protein: DP04167 (UniProt Q8N6T7)
Region: residues 1	to 25
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The cryo EM structure of the Nucleosome Bound SIRT6 shows this region lacks electron density, indicating it is disordered.
Cross-references: PDB:8OF4
EMDB:16845
Protein: DP04167 (UniProt Q8N6T7)
Region: residues 62	to 84
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The cryo EM structure of the Nucleosome Bound SIRT6 shows this region lacks electron density, indicating it is disordered.
Cross-references: PDB:8OF4
EMDB:16845
Protein: DP04167 (UniProt Q8N6T7)
Region: residues 293	to 355
Annotation: nucleosome binding (Molecular function)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
Thus, we introduced truncations of varying lengths at the C-terminus and evaluated these variants for nucleosome binding (Fig. 3b). The C-terminal deletion mutants exclusively formed 2:1 SIRT6:NCP complexes in the native gel without a detectable 1:1 intermediate, suggesting that the high affinity site was compromised, which led to a similar affinity for both binding events. As a result, the overall KD of SIRT6(1–292) and SIRT6(1–301) (KD = 254 and 139 nM, respectively) to nucleosomes is an order of magnitude weaker than full-length SIRT6 (Fig. 3c, Supplementary Fig. 4b, and Table 1). Thus, the C-terminally truncated mutants display impaired binding to NCPs, as well as a loss of the asymmetric binding mechanism.
The CTD of SIRT6 is required for high-affinity binding
Under this model, binding to the lower affinity site (KD(Low)) would be similar between SIRT6 and SIRT6(1–292). To test this mechanism, we first saturated the high affinity site on NCPs with 10 nM full-length SIRT6, then added increasing concentrations of SIRT6(1–292) and subjected the resulting complexes to native gel analysis (Fig. 3d and Table 1). The data revealed a dissociation constant (40 nM) similar to the KD(Low) value reported by the FRET experiment (Fig. 1e), supporting a model in which the CTD contributes to only the higher affinity binding event.
Protein: DP04167 (UniProt Q8N6T7)
Region: residues 330	to 348
Annotation: nucleosome binding (Molecular function)
Evidence: direct assay evidence used in manual assertion
Supporting statements:
To further interrogate this mechanism, we used an antibody raised against SIRT6 C-terminal residues 330–348 as a probe in EMSA experiments. The antibody was allowed to bind full-length SIRT6 before nucleosome addition (Supplementary Fig. 4c). When only the 1:1 SIRT6:NCP complex is apparent at 20 nM SIRT6, the antibody fully disrupted this complex (lanes 3 and 4). When both the 1:1 and 2:1 complexes appeared at 100 nM SIRT6, the antibody displaced part of the bound population while supershifting the rest (lanes 5 and 6). Finally, when both binding sites were saturated, only supershifting occurred, likely due to CTD-independent interactions driving binding at higher SIRT6 concentrations (lanes 8 and 9). Collectively, the results are consistent with the observation that the CTD is not necessary for complete SIRT6:NCP assembly (Fig. 3b), yet is essential for tight and multivalent binding at a single, high affinity site.
Protein: DP04167 (UniProt Q8N6T7)
Region: residues 293	to 355
Annotation: DNA binding (Molecular function)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
In EMSA experiments using 500 nM protein incubated with the 601-positioning DNA sequence, full-length SIRT6 displayed highest affinity for DNA, while the mutants bound noticeably weaker (Fig. 4a).
The CTD is an intrinsically disordered DNA-binding domain
Protein: DP04167 (UniProt Q8N6T7)
Region: residues 270	to 355
Annotation: disorder (Structural state)
Evidence: proton-based nuclear magnetic resonance evidence used in manual assertion
Supporting statements:
To confirm that the CTD directly interacts with DNA, we expressed and purified the isolated domain (residues 270–355) and evaluated DNA binding capacity (Supplementary Fig. 1a).
To confirm these predictions, we determined the 1H NMR spectrum of the CTD and observed peak clustering at ~7.0–8.5 ppm, which is characteristic of intrinsically disordered domains (Fig. 4b).
Protein: DP04167 (UniProt Q8N6T7)
Region: residues 270	to 355
Annotation: DNA binding (Molecular function)
Evidence: electrophoretic mobility shift assay evidence used in manual assertion
Supporting statements:
In EMSA experiments, we found that the CTD bound random sequences of DNA in a commercial 10 base pair ladder, as well as the 601-positioning sequence (Fig. 4c and Supplementary Fig. 5c). More strikingly, binding to the latter sequence with and without the histone octamer assembled on it produced exact binding isotherms. This reveals that the CTD is not only indiscriminate of DNA sequences, but recognizes both linear DNA as well as the curved, distorted DNA inherent in nucleosomes.
Protein: DP04167 (UniProt Q8N6T7)
Region: residues 270	to 355
Annotation: histone deacetylase activity (H3-K9 specific) (Molecular function)
Evidence: high-performance liquid chromatography evidence used in manual assertion
Supporting statements:
Our binding studies above predict that full-length SIRT6 would be a more efficient enzyme than a counterpart without the CTD. Indeed, when provided with MNase-digested nucleosomes as substrate, the full-length enzyme efficiently deacetylated endogenous H3K9ac, while SIRT6(1–292) and SIRT6(1–301) deacetylated the modification much slower (Fig. 5b and Supplementary Fig. 6a). Therefore, efficient chromatin deacetylation requires the CTD-dependent nucleosome-binding mechanism.
The CTD promotes efficient chromatin deacetylation by SIRT6
Protein: DP04167 (UniProt Q8N6T7)
Region: residues 293	to 344
Annotation: chromatin binding (Molecular function)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
To analyze the sub-cellular distribution of the SIRT6 constructs, we fractionated HCT116 cells into cytoplasmic and nucleoplasmic fractions. Compared to wild-type SIRT6, SIRT6Δ293–344 and SIRT6Δ302–344 levels were higher in both fractions (Supplementary Fig. 6b), suggesting that truncating the CTD led to weakened chromatin association.
Protein: DP04167 (UniProt Q8N6T7)
Region: residues 270	to 355
Annotation: nucleosomal DNA binding (Molecular function)
Evidence: electrophoretic mobility shift assay evidence used in manual assertion
Supporting statements:
In EMSA experiments, we found that the CTD bound random sequences of DNA in a commercial 10 base pair ladder, as well as the 601-positioning sequence (Fig. 4c and Supplementary Fig. 5c). More strikingly, binding to the latter sequence with and without the histone octamer assembled on it produced exact binding isotherms. This reveals that the CTD is not only indiscriminate of DNA sequences, but recognizes both linear DNA as well as the curved, distorted DNA inherent in nucleosomes.
Protein: DP04167 (UniProt Q8N6T7)
Region: residues 290	to 355
Annotation: protein import into nucleus (Biological process)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
Conversely, progressive deletion of the CTE of SIRT6 (ΔC2 and ΔC) resulted in a partial or dramatic mislocalization of SIRT6 from the nucleus to the cytoplasm (Figure 2B).
The ΔC2 construct comprises the region 1-289 and ΔC the 1-276 region.
Protein: DP04167 (UniProt Q8N6T7)
Region: residues 345	to 351
Annotation: protein import into nucleus (Biological process)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
Inspection of the C-terminal sequence of SIRT6 identified a seven-amino acid sequence starting at residue 345—345PKRVKAK351—that resembles a canonical nuclear localization signal (NLS). We mutated the four basic residues (K346, R347, K349, K351) of this putative NLS to alanine in the context of the full-length protein (to generate SIRT6-mutNLS) and examined its localization. GFP-tagged SIRT6-mutNLS was partially mislocalized to the cytoplasm (Figure 2B), and this pattern was indistinguishable from that generated by deleting ∼80 amino acids of the C terminus (ΔC2). These data suggest that the 345PKRVKAK351 sequence at the C terminus of SIRT6 is an NLS essential for proper nuclear localization.
The ΔC2 construct comprises the region 1-289 and ΔC the 1-276 region.