LiteFold/DisProt huggingface dataset:
Protein: DP02551 (UniProt Q8NBP7)
Region: residues 31	to 53
Annotation: self-inhibition (Disorder function)
Evidence: combinatorial experimental and author inference evidence contained in single publication used in manual assertion
Supporting statements:
the deletion of the acidic stretch creates a hyperactive PCSK9 displaying enhanced cellular uptake, increased potency of LDLR degradation, and stronger binding to LDLR [8], [37], [46]. This indicates that the acidic stretch has auto-inhibitory activity and functions as a negative regulator of PCSK9. The auto-inhibitory activity is not mediated by specific structural motifs. The acidic stretch could be shortened by a few residues, and up to six glutamic or aspartic acid residues could be charge-neutralized without changes in the auto-inhibitory activity, indicating that activity only depends on a minimal length and a minimal net charge [46].
Protein: DP02551 (UniProt Q8NBP7)
Region: residues 36	to 45
Annotation: disorder to order (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Solution NMR experiments with peptides spanning the acidic stretch region demonstrated a propensity to adopt an α-helix.
The region with α-helical propensity detected by NMR is a close match to the α-helical segment observed in the crystal structures (37DYEELVLAL45)
Protein: DP02551 (UniProt Q8NBP7)
Region: residues 36	to 45
Annotation: disorder to order (Structural transition)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
This helical conformation is stabilized by binding to antibody 6E2 (Ab6E2), as demonstrated by crystal structures of Fab6E2–peptide complexes. This antibody, which specifically recognizes the helical conformation, bound to both acidic stretch-derived peptides and to intact PCSK9, suggesting that the disorder-to-order transition is a physiological property of PCSK9.
The structures of the complexes of Fab6E2–Pep1 (wild-type), Fab6E2–Pep4 (sY38:pS47), and Fab6E2–Pep6 (E32K) were determined at 2.25, 2.20, and 2.10 Å resolution, respectively (see X-ray metrics in Suppl. Table S1). Overall, these complexes are remarkably similar (Fig. 2a–c). The most striking feature in all structures is a well-ordered 2 1/4-turn α-helix formed by the 9-amino-acid central portions of the peptides (37DYEELVLAL45).
Cross-references: PDB:6E4Y
PDB:6E4Z
Protein: DP02551 (UniProt Q8NBP7)
Region: residues 31	to 53
Annotation: protein binding (Molecular function)
Evidence: surface plasmon resonance evidence used in manual assertion
Supporting statements:
Immunoblotting of conditioned media demonstrated that Ab6E2 specifically recognized the prodomain of PCSK9 secreted by the three cell lines (Fig. 1a). Furthermore, experiments with the N-terminally truncated PCSK9, in which the acidic stretch residues Q31–A53 were deleted (PCSK9Δ53), indicated that the Ab6E2 binding site is located within the acidic stretch region. First, Ab6E2 did not detect the prodomain band of PCSK9Δ53 in immunoblotting experiments (Fig. 1a). Second, surface plasmon resonance (SPR) experiments demonstrated that Ab6E2 bound to PCSK9 with a KD of 10.5 ± 1.2 nM, but did not interact with PCSK9Δ53 (Fig. 1b, inset).
To investigate whether the region deleted in PCSK9Δ53 (Q31–A53) contains the main antibody epitope, we synthesized a peptide corresponding to this sequence, except that the N-terminal Q31 was omitted. This peptide (Pep1) bound to Ab6E2 with a KD value of 3.8 μM (Table 1), confirming that the antibody epitope lies within the acidic stretch.
Remarkably, Pep2 with the sY38 modification had an approximately 2000-fold increased affinity to Ab6E2 (KD = 1.9 nM)
Protein: DP02551 (UniProt Q8NBP7)
Region: residues 36	to 40
Annotation: molecular recognition display site (Disorder function)
Evidence: combinatorial experimental and author inference evidence contained in single publication used in manual assertion
Supporting statements:
The Y38 residue undergoes O-sulfation mediated by the enzyme sulfotransferase [32,33].
Protein: DP02551 (UniProt Q8NBP7)
Region: residues 37	to 45
Annotation: molecular function regulator (Molecular function)
Evidence: combinatorial experimental and author inference evidence contained in single publication used in manual assertion
Supporting statements:
There is strong evidence that the acidic stretch is involved in binding interactions. A recent study has implicated the acidic stretch in PCSK9 binding to LDL particles, which seems to regulate the activity of PCSK9 towards LDLR [45].
Protein: DP02552 (UniProt P43629)
Region: residues 361	to 444
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Consistent with the computer prediction that 3DL1-cyto is disordered, the1H-15N HSQC spectrum (Figure 1B) exhibits limited chemical shift dispersion in the NH dimension (8–8.6ppm).
Cross-references: BMRB:27651
Protein: DP02552 (UniProt P43629)
Region: residues 371	to 385
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The CON spectra of unphosphorylated SET-3DL1-cyto complexes with each individual SH2-domain (inmolar ratios of SET-3DL1:N-SH2 or C-SH2 at 1:0.2) show binding-induced reduction of intensities for peaks mainly from residues at or surrounding the N-ITIM (371-DPEEVTYAQLPHCVF-385).
The NMR spectrum of the complex with N-SH2 also shows minor peak intensity reductions for residues 400-PPTDTILYTELPN-412 spanning the C-terminal ITIM (Figures 7A, black andS3A, black to red) that are not evident in complex with C-SH2
Cross-references: BMRB:27651
Protein: DP02552 (UniProt P43629)
Region: residues 375	to 379
Annotation: phosphorylation display site (Disorder function)
Evidence: combinatorial experimental and author inference evidence contained in single publication used in manual assertion
Supporting statements:
3DL1binding to MHC-I ligand results in changes in local electrostatic interactions surrounding the TM and membrane-proximal region of 3DL1-cyto, which induces conformational changes in the N-terminal membrane-proximal region, including loss of transient ahelices, and hence liberating tyrosines from the membrane (Figures 8B and 8C) to be accessible for interaction with the N-SH2 of SHP-2 (Figure 8B) and phosphorylation by Src family PTK (Figure 8C). Phosphorylation of both ITIM tyrosines facilitates high-affinity interaction with both SH2 domains of SHP-2(or SHP-1) to fully activate these PTPs at the plasma membrane.
Protein: DP02552 (UniProt P43629)
Region: residues 361	to 444
Annotation: molecular adaptor activity (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
3DL1 binding to MHC-I ligand results in changes in local electrostatic interactions surrounding the TM and membrane-proximal region of 3DL1-cyto, which induces conformational changes in the N-terminal membrane-proximal region, including loss of transient a-helices, and hence liberating tyrosines from the membrane (Figures 8B and 8C) to be accessible for interaction with the N-SH2 of SHP-2 (Figure 8B) and phosphorylation by Src family PTK (Figure 8C). Phosphorylation of both ITIM tyrosines facilitates high-affinity interaction with both SH2 domains of SHP-2(or SHP-1) to fully activate these PTPs at the plasma membrane.
Protein: DP02552 (UniProt P43629)
Region: residues 405	to 409
Annotation: phosphorylation display site (Disorder function)
Evidence: combinatorial experimental and author inference evidence contained in single publication used in manual assertion
Supporting statements:
3DL1binding to MHC-I ligand results in changes in local electrostatic interactions surrounding the TM and membrane-proximal region of 3DL1-cyto, which induces conformational changes in the N-terminal membrane-proximal region, including loss of transient ahelices, and hence liberating tyrosines from the membrane (Figures 8B and 8C) to be accessible for interaction with the N-SH2 of SHP-2 (Figure 8B) and phosphorylation by Src family PTK (Figure 8C). Phosphorylation of both ITIM tyrosines facilitates high-affinity interaction with both SH2 domains of SHP-2(or SHP-1) to fully activate these PTPs at the plasma membrane.
Protein: DP02554 (UniProt Q6P5W5)
Region: residues 424	to 498
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The 1H‐15N HSQC spectrum of hZIP4 ICL2 measured at 10°C [Fig. 1(c)] displays a narrow chemical shift dispersion in the 1H dimension ranging from 7.8 to 8.8 ppm. Reduced chemical shift dispersion in the 1H dimension is characteristic of IDRs compared to spectra for folded proteins.
Values for secondary chemical shifts were small (Supporting Information Fig. S3), suggesting that the ICL2 protein region exists predominantly as a random coil. Transient secondary structure populations for the disordered hZIP4 ICL2 were assessed using both the secondary structure propensity (SSP) score41 and the δ2D method.42 Both methods (Fig. 2) indicated that the ICL2 protein region is largely random coil with slight tendencies for regions of β‐strand between residues 460–469 and 484–490.
Protein: DP02554 (UniProt Q6P5W5)
Region: residues 431	to 448
Annotation: metal ion binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Previously, the hZIP4 ICL2 was shown to coordinate two zinc ions with the first zinc binding at a CysHis3 site and the second zinc binding at a His4 site.19 The single cysteine residue (C436) was responsible for the nanomolar binding affinity of zinc to ICL2.
The 1H chemical shift peak dispersion of zinc‐bound ICL2 remained narrow, indicating that the protein remained disordered upon zinc binding. The most prominent change in the spectra upon zinc addition was the disappearance of cross‐peaks. Analysis of the peak heights [Fig. 3(b)] revealed a metal‐dependent signal broadening with cross‐peaks for residues in the histidine‐rich region (residues 431–448), exhibiting ~50% reduction in signal with one zinc bound and complete signal broadening in the presence of two molar equivalents of zinc.
Protein: DP02554 (UniProt Q6P5W5)
Region: residues 431	to 448
Annotation: zinc ion sequestering activity (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The largest intracellular loop of ZIP4, ICL2, regulates the plasma membrane levels of the transporter in response to intracellular zinc levels.18 At low intracellular zinc concentrations, ZIP4 undergoes endocytosis from the plasma membrane, and as zinc concentrations increase, ZIP4 is ubiquitinated at the single lysine residue (K463) and proteolytically degraded.
The 1H chemical shift peak dispersion of zinc‐bound ICL2 remained narrow, indicating that the protein remained disordered upon zinc binding. The most prominent change in the spectra upon zinc addition was the disappearance of cross‐peaks. Analysis of the peak heights [Fig. 3(b)] revealed a metal‐dependent signal broadening with cross‐peaks for residues in the histidine‐rich region (residues 431–448), exhibiting ~50% reduction in signal with one zinc bound and complete signal broadening in the presence of two molar equivalents of zinc.
Protein: DP02554 (UniProt Q6P5W5)
Region: residues 424	to 498
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Circular dichroism spectroscopy revealed that the M3M4 domain is intrinsically disordered, with only a small structural change induced upon Zn(2+) coordination.
Protein: DP02554 (UniProt Q6P5W5)
Region: residues 436	to 448
Annotation: zinc ion sequestering activity (Molecular function)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
Using a combination of site-directed mutagenesis, metal binding affinity assays, and X-ray absorption spectroscopy, we demonstrated that the two Zn(2+) ions bind sequentially, with the first Zn(2+) binding to a CysHis3 site with a nanomolar binding affinity, and the second Zn(2+) binding to a His4 site with a weaker affinity.
Our data supports a model in which the intracellular M3M4 domain senses high cytosolic Zn(2+) concentrations and regulates the plasma membrane levels of the hZIP4 transporter in response to Zn(2+) binding.
Protein: DP02554 (UniProt Q6P5W5)
Region: residues 436	to 448
Annotation: zinc ion binding (Molecular function)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
Using a combination of site-directed mutagenesis, metal binding affinity assays, and X-ray absorption spectroscopy, we demonstrated that the two Zn(2+) ions bind sequentially, with the first Zn(2+) binding to a CysHis3 site with a nanomolar binding affinity, and the second Zn(2+) binding to a His4 site with a weaker affinity.
Our data supports a model in which the intracellular M3M4 domain senses high cytosolic Zn(2+) concentrations and regulates the plasma membrane levels of the hZIP4 transporter in response to Zn(2+) binding.
Protein: DP02555 (UniProt Q15109)
Region: residues 364	to 404
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The 2D 1H–15N HSQC spectra of the peptide show a poor spreading of the signals, as commonly observed for unfolded proteins
Secondary structure prediction was carried out with the program TALOS+ (Shen et al. 2009) using the HN, N, C′, Cα, Hα and Cβ chemical shift values as input data. The prediction suggests a random-coil conformation for the whole sequence with all the residues experiencing high mobility. Further and more detailed information on the dynamics of cytRAGE were provided by relaxation data. In particular, the characterization of fast motions occurring on picosecond-nanosecond time scales was performed by exploiting the spin relaxation properties of the amide 15N nuclei through R1, R2, and NOE experiments (see Fig. 3a, c, e). Random-coil polypeptides and flexible protein regions are characterized by fast local motions with NOE values below 0.5 or negative. In our construct of cytRAGE, that includes also three aminoacids of the transmembrane region and two of the thrombin cleavage site, the heteronuclear NOEs for all amino acids are close to zero or even negative at the C-terminal region (Fig. 3e). These NOE values indicate that all the isolated cytRAGE is highly flexible and unfolded in solution.
Protein: DP02555 (UniProt Q15109)
Region: residues 364	to 404
Annotation: protein binding (Molecular function)
Evidence: combinatorial experimental and author inference evidence contained in single publication used in manual assertion
Supporting statements:
cytRAGE is reported to bind different intracellular proteins such as mDia-1(Rai et al. 2012), ERK-1/2 (Ishihara et al. 2003) and after phosphorylation by PKCξ, TIRAP and MyD88 (Sakaguchi et al. 2011).
Protein: DP02555 (UniProt Q15109)
Region: residues 364	to 404
Annotation: molecular adaptor activity (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The cytoplasmic domain of RAGE (C-terminal RAGE; ctRAGE) is critical for RAGE-dependent signal transduction. As the most membrane-proximal event, mDia1 binds to ctRAGE, and it is essential for RAGE ligand-stimulated phosphorylation of AKT and cell proliferation/migration. We show that ctRAGE contains an unusual α-turn that mediates the mDia1-ctRAGE interaction and is required for RAGE-dependent signaling.
Cross-references: PDB:2LMB
Protein: DP02555 (UniProt Q15109)
Region: residues 364	to 370
Annotation: protein binding (Molecular function)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
When Arg-5 and Gln-6 are mutated to alanines, the loss of the binding epitope for mDia1 suppresses RAGE ligand-induced downstream phosphorylation of AKT and migration and proliferation of vascular smooth muscle cells (SMCs).
To confirm the FH1-ctRAGE interaction surface, we made a ctRAGE double mutant in which residues Arg-5 and Gln-6 were changed to alanine to create R5A/Q6A-ctRAGE (Fig. 1A). Titrating unlabeled FH1 into [U-15N]R5A/Q6A-ctRAGE resulted only in small (<0.02 ppm) changes in the position of one amino acid residue, Arg-10. No other chemical shift changes or substantial broadening of the R5A/Q6A-ctRAGE peaks were observed, suggesting that the double mutant interacts with FH1 very weakly, with a KD well above 1 mm
Protein: DP02555 (UniProt Q15109)
Region: residues 364	to 404
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The 15N HSQC spectrum of ctRAGE is well resolved but has limited chemical shift dispersion (Fig. 1B), indicating that the majority of the ctRAGE residues are disordered.
Protein: DP02555 (UniProt Q15109)
Region: residues 364	to 370
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Titrating unlabeled FH1 into uniformly 15N-labeled ctRAGE, [U-15N]ctRAGE, resulted in specific changes in the HSQC spectrum of ctRAGE
These changes suggest that residues Gln-3 through Gln-6 participate in the interaction with FH1. Peak broadening is characteristic of slow to intermediate exchange and suggests that the dissociation constant of the interaction Kd is <10 μm
Protein: DP02555 (UniProt Q15109)
Region: residues 389	to 393
Annotation: phosphorylation display site (Disorder function)
Evidence: protein kinase assay evidence used in manual assertion
Supporting statements:
the cytoplasmic domain of RAGE is phosphorylated at Ser391 by PKCζ upon binding of ligands. TIRAP and MyD88, which are known to be adaptor proteins for Toll-like receptor-2 and -4 (TLR2/4), bound to the phosphorylated RAGE and transduced a signal to downstream molecules.
Protein: DP02555 (UniProt Q15109)
Region: residues 364	to 404
Annotation: molecular adaptor activity (Molecular function)
Evidence: combinatorial experimental and author inference evidence contained in single publication used in manual assertion
Supporting statements:
cytRAGE is reported to bind different intracellular proteins such as mDia-1(Rai et al. 2012), ERK-1/2 (Ishihara et al. 2003) and after phosphorylation by PKCξ, TIRAP and MyD88 (Sakaguchi et al. 2011).
Protein: DP02556 (UniProt P09077)
Region: residues 298	to 331
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
We have completely assigned the 1H, 13C, and 15N chemical shifts of the backbone atoms of SCRK298–K384 and DFDT337–K426 by solution NMR spectroscopy. Chemical shift-based secondary structure prediction and 15N dynamic studies show that ~ 30 residues in the N-terminus are completely disordered, while the DNA-binding homeodomain is properly folded in both proteins. Reduced spectral density analysis of the 15N dynamics data revealed variations in flexibility in the disordered region.
The N-terminal residues of both proteins (SCR: K298 to Y331; DFD: T337 to T374) show lack of secondary structure, highlighting their highly flexible nature.
Cross-references: BMRB:27622
Protein: DP02556 (UniProt P09077)
Region: residues 305	to 310
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Interestingly, one of these segments of rigid residues is conserved in both SCR and DFD, and using NMR titration experiments, we show that this rigid segment is specifically recognized by a co-transcription factor Extradenticle (EXD).
Unlabeled homeodomain of EXDA238–I300 (residues A238 toI300) was titrated into15N-labeled SCRK298–K384 or DFDT337–K426, and each titration point was monitored by 15N–1H HSQC experiment (Figs. 6and S4). Consecutive residues Y305 to R310 (P306 lacks backbone NH) in the rigid segment of SCRK298–K384 showed significant chemical shift changes
Cross-references: BMRB:27622
Protein: DP02556 (UniProt P09077)
Region: residues 305	to 310
Annotation: molecular function regulator (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Interestingly, one of these segments of rigid residues is conserved in both SCR and DFD, and using NMR titration experiments, we show that this rigid segment is specifically recognized by a co-transcription factor Extradenticle (EXD).
Unlabeled homeodomain of EXDA238–I300 (residues A238 toI300) was titrated into15N-labeled SCRK298–K384 or DFDT337–K426, and each titration point was monitored by 15N–1H HSQC experiment (Figs. 6and S4). Consecutive residues Y305 to R310 (P306 lacks backbone NH) in the rigid segment of SCRK298–K384 showed significant chemical shift changes
For SCR, it has been shown that the homeodomain and its preceding~30 residues are sufficient to carry out most of its in vivo functions such as homeotic transformations,transcriptional regulation, and protein–protein interactions[14]. Moreover, these N-terminal 30 residues have been shown to regulate the functional specificities in both SCR and DFD [15].
Cross-references: BMRB:27622
Protein: DP02557 (UniProt P07548)
Region: residues 337	to 374
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
We have completely assigned the 1H, 13C, and 15N chemical shifts of the backbone atoms of SCRK298–K384 and DFDT337–K426 by solution NMR spectroscopy. Chemical shift-based secondary structure prediction and 15N dynamic studies show that ~ 30 residues in the N-terminus are completely disordered, while the DNA-binding homeodomain is properly folded in both proteins. Reduced spectral density analysis of the 15N dynamics data revealed variations in flexibility in the disordered region.
The N-terminal residues of both proteins (SCR: K298 to Y331; DFD: T337 to T374) show lack of secondary structure, highlighting their highly flexible nature.
Cross-references: BMRB:27621
Protein: DP02557 (UniProt P07548)
Region: residues 341	to 349
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Interestingly, one of these segments of rigid residues is conserved in both SCR and DFD, and using NMR titration experiments, we show that this rigid segment is specifically recognized by a co-transcription factor Extradenticle (EXD).
Similarly, the stretch of residues R341 to K349 (P345 lacks backbone NH) in the rigid segment of DFDT337–K426 showed significant chemical shift changes (Fig. 6b). The remaining residues of both SCRK298–K384 and DFDT337–K426 have no significant chemical shift perturbation, indicating a very specific interaction between the rigid segment of the HOX proteins and EXD
Cross-references: BMRB:27621
Protein: DP02557 (UniProt P07548)
Region: residues 341	to 349
Annotation: molecular adaptor activity (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Interestingly, one of these segments of rigid residues is conserved in both SCR and DFD, and using NMR titration experiments, we show that this rigid segment is specifically recognized by a co-transcription factor Extradenticle (EXD).
Similarly, the stretch of residues R341 to K349 (P345 lacks backbone NH) in the rigid segment of DFDT337–K426 showed significant chemical shift changes (Fig. 6b). The remaining residues of both SCRK298–K384 and DFDT337–K426 have no significant chemical shift perturbation, indicating a very specific interaction between the rigid segment of the HOX proteins and EXD
Cross-references: BMRB:27621