LiteFold/DisProt huggingface dataset:
Protein: DP04401 (UniProt A0AAI8ESY0)
Region: residues 154	to 255
Annotation: order (Structural state)
Evidence: protein thermal shift assay evidence used in manual assertion
Supporting statements:
Notably, the thermal denaturation experiment showed that the complex has significantly higher unfolding enthalpy (29.57 kcal/mol) than the unbound VirB9Ct (10.42 kcal/mol; Figure 2C). This observation may be explained considering that VirB9Ct−VirB7Nt unfolding and dissociation
occur within a single transition as a consequence of the coupling between these two processes.
Protein: DP04401 (UniProt A0AAI8ESY0)
Region: residues 154	to 255
Annotation: molten globule to order (Structural transition)
Evidence: protein thermal shift assay evidence used in manual assertion
Supporting statements:
Notably, the thermal denaturation experiment showed that the complex has significantly higher unfolding enthalpy (29.57 kcal/mol) than the unbound VirB9Ct (10.42 kcal/mol; Figure 2C). This observation may be explained considering that VirB9Ct−VirB7Nt unfolding and dissociation
occur within a single transition as a consequence of the coupling between these two processes.
Protein: DP04401 (UniProt A0AAI8ESY0)
Region: residues 154	to 255
Annotation: protein binding (Molecular function)
Evidence: isothermal titration calorimetry evidence used in manual assertion
Supporting statements:
Binding of VirB9Ct to VirB7Nt is enthalpically driven as indicated by isothermal titration calorimetry (ITC) experiments carried out at 10, 25, and 35 °C (Figure 3A,B). The VirB9Ct−VirB7Nt Kd app (see Experimental Section) is 0.70 μM at 35 °C, similar to the value reported before.
Protein: DP04401 (UniProt A0AAI8ESY0)
Region: residues 154	to 171
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The backbone 15N chemical shifts for residues 163−171 along the β1 showed a very good agreement with random coil chemical shifts predicted from the VirB9Ct amino acid sequence using the ncIDP online server, while a poorer agreement was observed along β2 (Figure 6A,B), suggesting that the VirB9Ct β1 is disordered in the absence of VirB7Nt.
The authors already stated in their earlier article (PMID:27594685) that the S2 order parameters of the termini of the VirB9Ct domain are low, so they are disodered (even in the complex), although residue boundaries were not mentioned, so the regions could not be annotated based on that statement. Here they show that in the unbound state the first beta strand, β1, is disodered, which ultimately means that the whole N-terminal region up until residue 171 must be disordered. This is further supported by the H−15N HSQC spectrum of unbound VirB9Ct in Figure 4A, where all the resolved peaks belonging to residues 154-171 show a narrow peak distribution around 8.5 pmm that is typical for disordered regions.
NMR data obtained for VirB9Ct in the unbound state at 35 °C showed strong evidence that β1 is unstructured in the absence of VirB7Nt, while
other residues also located at the VirB7Nt binding region experience an equilibrium between multiple conformations at the millisecond time scale.
Cross-references: BMRB:51836
BMRB:52005
Protein: DP04401 (UniProt A0AAI8ESY0)
Region: residues 154	to 255
Annotation: protein binding (Molecular function)
Evidence: stopped-flow fluorescence spectroscopy evidence
Supporting statements:
The presence of tryptophan residues at positions 177 and 34 in VirB9Ct and VirB7Nt, respectively, and the millisecond exchange time scale
between unbound and bound VirB9Ct states, favored the use of stopped-flow fluorescence to follow the VirB9Ct−VirB7Nt association kinetics.
These observations suggest that the VirB9Ct interaction with VirB7Nt could be described by a conformational-selection mechanism at 25 °C.
Protein: DP04401 (UniProt A0AAI8ESY0)
Region: residues 154	to 255
Annotation: disorder (Structural state)
Evidence: heteronuclear single quantum coherence spectroscopy evidence used in manual assertion
Supporting statements:
The 15N-HSQC spectrum of VirB9XAC2620_154–255 showed characteristics of poor line shape and chemical shift dispersion (Figure 4; red) suggestive of conformational disorder and a probable lack of stable tertiary structure.
Protein: DP04401 (UniProt A0AAI8ESY0)
Region: residues 154	to 255
Annotation: type IV secretion system complex (Cellular component)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
This analysis showed that only residues 27–41, within the disordered VirB7 N-terminus, undergo significant chemical shift perturbations (Figure 3C and D). This region is adjacent to, but does not overlap, the N-terminal region involved in VirB7XAC2622 oligomerization (residues 42–49).
Chemical shift perturbation data showed that two regions are involved in VirB7XAC2622 self-interactions: a region in the unfolded N-terminus (residues 42–49) and a patch on the surface of the globular domain made up of residues 63–65, 85–93, 111–119 and 131 (Figure 2B and C).
In well-characterized T4SSs, VirB7 is a lipoprotein attached to the periplasmic side of the outer membrane [28]. It has been shown to interact with several T4SS subunits [29], including itself [30] and the VirB9 C-terminal region [31], [32].
Protein: DP04401 (UniProt A0AAI8ESY0)
Region: residues 154	to 255
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
In order to map the VirB9 binding site on the structure of VirB7, we assigned the backbone resonances of VirB7XAC2622_24–139 in complex with VirB9XAC2620_154–255 and analyzed the chemical shift differences (Figure 3A and B). This analysis showed that only residues 27–41, within the disordered VirB7 N-terminus, undergo significant chemical shift perturbations (Figure 3C and D).
Protein: DP04416 (UniProt O53692)
Region: residues 1	to 14
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The core of the complex is well defined (residues 15–79 in EsxG and 17–75 in EsxH), although there appears to be significant flexibility in the hairpin loop of EsxG (residues 40–47) as well as the N and C termini of EsxG and EsxH, which form flexible arms at both ends of the complex (Fig. 1).
Cross-references: PDB:2KG7
Protein: DP04416 (UniProt O53692)
Region: residues 1	to 14
Annotation: flexible N-terminal tail (Disorder function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The core of the complex is well defined (residues 15–79 in EsxG and 17–75 in EsxH), although there appears to be significant flexibility in the hairpin loop of EsxG (residues 40–47) as well as the N and C termini of EsxG and EsxH, which form flexible arms at both ends of the complex (Fig. 1).
Cross-references: PDB:2KG7
Protein: DP04416 (UniProt O53692)
Region: residues 80	to 97
Annotation: flexible C-terminal tail (Disorder function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The core of the complex is well defined (residues 15–79 in EsxG and 17–75 in EsxH), although there appears to be significant flexibility in the hairpin loop of EsxG (residues 40–47) as well as the N and C termini of EsxG and EsxH, which form flexible arms at both ends of the complex (Fig. 1).
Cross-references: PDB:2KG7
Protein: DP04416 (UniProt O53692)
Region: residues 80	to 97
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The core of the complex is well defined (residues 15–79 in EsxG and 17–75 in EsxH), although there appears to be significant flexibility in the hairpin loop of EsxG (residues 40–47) as well as the N and C termini of EsxG and EsxH, which form flexible arms at both ends of the complex (Fig. 1).
Cross-references: PDB:2KG7
Protein: DP04423 (UniProt P0DXN6)
Region: residues 400	to 525
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The very small spread of the resonance frequencies for amide protons (between 7.8 and 8.7 ppm; see frame in Fig.2A) together with the scarcity of nuclear Overhauser effects in the amide-amide region are typical of a protein without any stable secondary structure.
Protein: DP04423 (UniProt P0DXN6)
Region: residues 400	to 525
Annotation: disorder (Structural state)
Evidence: chromatography evidence used in manual assertion
Supporting statements:
In the case of NTAIL, RSN = 19 Å and RSU = 35 Å. Therefore, the Stokes radius experimentally measured for NTAIL (27 Å) is not compatible with a monomeric, globular protein. Rather, such a large value of the Stokes radius can be attributed either to dimerization or to an extended conformation.
Protein: DP04423 (UniProt P0DXN6)
Region: residues 401	to 525
Annotation: disorder (Structural state)
Evidence: atomic force microscopy evidence used in manual assertion
Supporting statements:
The IDR showed a temporarily appearing and disappearing small globule at the N terminus corresponding to Box1 (Fig. 4d). Except for this, the IDR was observed to be fully disordered (with mean height of 0.4−0.5 nm) (Extended Data Fig. 5a).
Protein: DP04423 (UniProt P0DXN6)
Region: residues 401	to 420
Annotation: disorder to molten globule (Structural transition)
Evidence: atomic force microscopy evidence used in manual assertion
Supporting statements:
The IDR showed a temporarily appearing and disappearing small globule at the N terminus corresponding to Box1 (Fig. 4d).
he H2 value of 0.8 nm is larger than the height of fully disordered regions, indicating that Box1 and Box2 are both partially ordered, even in their lower height states.
Measles virus (MeV) nucleoprotein Box 1 region is comprised within the residues 401–420
Protein: DP04423 (UniProt P0DXN6)
Region: residues 482	to 502
Annotation: disorder to molten globule (Structural transition)
Evidence: atomic force microscopy evidence used in manual assertion
Supporting statements:
The H2 value of 0.8 nm is larger than the height of fully disordered regions, indicating that Box1 and Box2 are both partially ordered, even in their lower height states.
Measles virus (MeV) nucleoprotein Box 2 region is comprised within the residues 482–502.
Protein: DP04423 (UniProt P0DXN6)
Region: residues 401	to 525
Annotation: molecular adaptor activity (Molecular function)
Evidence: mass spectrometry evidence used in manual assertion
Supporting statements:
The spectra are dominated by the peaks of the free proteins, but signals specific to the NTAIL–PXD complex become evident at the higher protein concentration. The measured mass of the complex (21,187.4 ± 0.61 Da) corresponds closely to the sum of the theoretical values expected for NTAIL with the initial methionine and PXD without the initial methionine (21,187.46 Da), indicating a 1:1 stoichiometry.
Protein: DP04423 (UniProt P0DXN6)
Region: residues 401	to 525
Annotation: protein binding (Molecular function)
Evidence: small-angle X-ray scattering evidence used in manual assertion
Supporting statements:
The molecular mass (MM) calculated from the forward scattering intensity I(0) is 23.5 ± 2 kDa, in agreement with the value expected for a 1:1 stoichiometric complex (21.3 kDa), thus suggesting that the complex did form in solution.
The high value of Rg indicated that the overall structure of the NTAILHN–XD complex is not compact.
Protein: DP04423 (UniProt P0DXN6)
Region: residues 401	to 516
Annotation: disorder (Structural state)
Evidence: sodium dodecyl sulfate polyacrylamide gel electrophoresis evidence used in manual assertion
Supporting statements:
As shown in Figure 3B, the three NTAIL deletion proteins migrate in SDS-PAGE with an apparent MM of either 20 kDa (NTAILΔ3 and NTAILΔ1) or 18 kDa (NTAILΔ2,3) (expected MMs are 14.5, 13.4, and 11.5 kDa, respectively). This abnormal migratory behavior has already been documented for NTAIL, where mass spectrometry analysis and N-terminal sequencing gave the expected results (Longhi et al. 2003). The anomalous electrophoretic mobility is therefore due to a rather high content of acidic residues, as frequently observed in intrinsically disordered proteins (Tompa 2002).
Protein: DP04423 (UniProt P0DXN6)
Region: residues 401	to 488
Annotation: disorder (Structural state)
Evidence: sodium dodecyl sulfate polyacrylamide gel electrophoresis evidence used in manual assertion
Supporting statements:
As shown in Figure 3B, the three NTAIL deletion proteins migrate in SDS-PAGE with an apparent MM of either 20 kDa (NTAILΔ3 and NTAILΔ1) or 18 kDa (NTAILΔ2,3) (expected MMs are 14.5, 13.4, and 11.5 kDa, respectively). This abnormal migratory behavior has already been documented for NTAIL, where mass spectrometry analysis and N-terminal sequencing gave the expected results (Longhi et al. 2003). The anomalous electrophoretic mobility is therefore due to a rather high content of acidic residues, as frequently observed in intrinsically disordered proteins (Tompa 2002).
Protein: DP04423 (UniProt P0DXN6)
Region: residues 421	to 525
Annotation: disorder (Structural state)
Evidence: sodium dodecyl sulfate polyacrylamide gel electrophoresis evidence used in manual assertion
Supporting statements:
As shown in Figure 3B, the three NTAIL deletion proteins migrate in SDS-PAGE with an apparent MM of either 20 kDa (NTAILΔ3 and NTAILΔ1) or 18 kDa (NTAILΔ2,3) (expected MMs are 14.5, 13.4, and 11.5 kDa, respectively). This abnormal migratory behavior has already been documented for NTAIL, where mass spectrometry analysis and N-terminal sequencing gave the expected results (Longhi et al. 2003). The anomalous electrophoretic mobility is therefore due to a rather high content of acidic residues, as frequently observed in intrinsically disordered proteins (Tompa 2002).
Protein: DP04423 (UniProt P0DXN6)
Region: residues 401	to 516
Annotation: disorder (Structural state)
Evidence: gel-filtration evidence used in manual assertion
Supporting statements:
As expected for intrinsically disordered protein subdomains, the Stokes radius (RS) values, as inferred by gel filtration (27 ± 3 Å, 22 ± 3 Å, and 27 ± 3 Å for NTAILΔ3, NTAILΔ2,3, and NTAILΔ1, respectively), are consistent with extended conformations (see Materials and Methods).
Protein: DP04423 (UniProt P0DXN6)
Region: residues 401	to 488
Annotation: disorder (Structural state)
Evidence: gel-filtration evidence used in manual assertion
Supporting statements:
As expected for intrinsically disordered protein subdomains, the Stokes radius (RS) values, as inferred by gel filtration (27 ± 3 Å, 22 ± 3 Å, and 27 ± 3 Å for NTAILΔ3, NTAILΔ2,3, and NTAILΔ1, respectively), are consistent with extended conformations (see Materials and Methods).
Protein: DP04423 (UniProt P0DXN6)
Region: residues 421	to 525
Annotation: disorder (Structural state)
Evidence: gel-filtration evidence used in manual assertion
Supporting statements:
As expected for intrinsically disordered protein subdomains, the Stokes radius (RS) values, as inferred by gel filtration (27 ± 3 Å, 22 ± 3 Å, and 27 ± 3 Å for NTAILΔ3, NTAILΔ2,3, and NTAILΔ1, respectively), are consistent with extended conformations (see Materials and Methods).
Protein: DP04423 (UniProt P0DXN6)
Region: residues 401	to 525
Annotation: disorder (Structural state)
Evidence: small-angle X-ray scattering evidence used in manual assertion
Supporting statements:
The high value of Rg indicated that the overall structure of the NTAILHN–XD complex is not compact. The distances distribution function inferred from the scattering curve of the NTAILHN–XD complex exhibits a maximum at 20 Å, with a shoulder at about 30 Å and a long tail up to 146 Å, typical of an elongated object (see Fig. 2A).
Protein: DP04423 (UniProt P0DXN6)
Region: residues 401	to 525
Annotation: protein binding (Molecular function)
Evidence: surface plasmon resonance evidence used in manual assertion
Supporting statements:
Binding affinities between XD and NTAIL constructs were established using 180–225 RU of immobilized XD and NTAIL concentrations ranging from 0.1 to 10 μM (see Materials and Methods). Dosage-dependent binding was observed in this range. Reactions conformed to a 1:1 ligand-substrate (Langmuir) binding model, exhibiting an excellent fit (i.e., a χ2 value <1 and residuals within the range of ±2) following global analysis of sensorgrams.
Protein: DP04423 (UniProt P0DXN6)
Region: residues 517	to 525
Annotation: protein binding (Molecular function)
Evidence: surface plasmon resonance evidence used in manual assertion
Supporting statements:
In contrast, removal of either Box3 alone or Box2 plus Box3 results in a strong decrease (three orders of magnitude) in the equilibrium dissociation constant, where NTAILΔ3 and NTAILΔ2,3 display similar binding affinities (see Table 1). The strong decrease in the affinity resulting from removal of Box3 clearly indicates that Box2 is not the sole region involved in binding to XD, and attests that Box3 also plays a role in the interaction with XD, as already suggested by SAXS studies.
Measles virus (MeV) nucleoprotein Box 3 region is comprised within the residues 517-525.
Protein: DP04423 (UniProt P0DXN6)
Region: residues 489	to 525
Annotation: protein binding (Molecular function)
Evidence: surface plasmon resonance evidence used in manual assertion
Supporting statements:
In contrast, removal of either Box3 alone or Box2 plus Box3 results in a strong decrease (three orders of magnitude) in the equilibrium dissociation constant, where NTAILΔ3 and NTAILΔ2,3 display similar binding affinities (see Table 1).
Measles virus (MeV) nucleoprotein Box 2 and Box 3 region is comprised within the residues 488-525.
Protein: DP04423 (UniProt P0DXN6)
Region: residues 401	to 516
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The far-UV circular dichroism (CD) spectra of NTAIL deletion proteins at neutral pH are typical of unstructured proteins, as seen by their large negative ellipticity at 198 nm and very low ellipticity at 185 nm (Fig. 4A–C).
Protein: DP04423 (UniProt P0DXN6)
Region: residues 401	to 488
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The far-UV circular dichroism (CD) spectra of NTAIL deletion proteins at neutral pH are typical of unstructured proteins, as seen by their large negative ellipticity at 198 nm and very low ellipticity at 185 nm (Fig. 4A–C).
Protein: DP04423 (UniProt P0DXN6)
Region: residues 421	to 525
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The far-UV circular dichroism (CD) spectra of NTAIL deletion proteins at neutral pH are typical of unstructured proteins, as seen by their large negative ellipticity at 198 nm and very low ellipticity at 185 nm (Fig. 4A–C).
Protein: DP04423 (UniProt P0DXN6)
Region: residues 401	to 525
Annotation: protein binding (Molecular function)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
After mixing NTAILHNFC with different molar excesses of XD, the observed CD spectra differed from the corresponding theoretical average curves calculated from the individual spectra.
Protein: DP04423 (UniProt P0DXN6)
Region: residues 401	to 525
Annotation: disorder to order (Structural transition)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Since the theoretical average curves correspond to the spectra that would be expected if no structural variations occur, deviations from these curves indicate structural transitions. The results obtained in the presence of a threefold molar excess of XD indicate a random coil to α-helix transition, as judged by the much more pronounced minima at 208 nm and 222 nm, and by the higher ellipticity at 190 nm of the experimentally observed spectrum compared to the corresponding theoretical average curve (see Fig. 5A).
Protein: DP04423 (UniProt P0DXN6)
Region: residues 401	to 516
Annotation: disorder to order (Structural transition)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Removal of Box3 significantly reduces, but does not abrogate, the folding ability of NTAIL. Indeed, the experimental CD spectrum does not significantly deviate from the average curve in the 200–260 nm region, but it considerably deviates from the average curve in the 185–195 region (58% mean increase of ellipticity) (Fig. 5B), thus supporting partial folding ability of NTAILΔ3 in the presence of XD.
Measles virus (MeV) nucleoprotein Box 3 region is comprised within the residues 517-525
Protein: DP04423 (UniProt P0DXN6)
Region: residues 421	to 525
Annotation: disorder to order (Structural transition)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Conversely, removal of Box1 does not affect the folding ability of NTAIL, as the deviations from the average spectrum are similar to those observed with the full-length form (Fig. 5, cf. D and A). The mean increase in ellipticity in the 185–195 nm region (77%) and the decrease in the ellipticity value at 220 nm (46%) observed with NTAILΔ1, are comparable to the corresponding values observed with NTAILHNFC (70% and 46%, respectively).
Measles virus (MeV) nucleoprotein Box 1 region is comprised within the residues 401-420.
Protein: DP04423 (UniProt P0DXN6)
Region: residues 401	to 416
Annotation: disorder to order (Structural transition)
Evidence: heteronuclear single quantum coherence spectroscopy evidence used in manual assertion
Supporting statements:
As shown in Figure 7, the same peaks undergoing the large displacement in the NTAILHN–XD complex are also observed in the NTAILΔ3–XD complex. In particular, among these peaks, the occurrence in both complexes of the 11 peaks that undergo the random coil to α-helix transition (see Fig. 7, stars), provides further support that the helical folding occurs within Box2.
Protein: DP04423 (UniProt P0DXN6)
Region: residues 401	to 525
Annotation: entropic chain (Disorder function)
Evidence: small-angle X-ray scattering evidence used in manual assertion
Supporting statements:
The high value of Rg indicated that the overall structure of the NTAILHN–XD complex is not compact. The distances distribution function inferred from the scattering curve of the NTAILHN–XD complex exhibits a maximum at 20 Å, with a shoulder at about 30 Å and a long tail up to 146 Å, typical of an elongated object (see Fig. 2A). The bump most probably corresponds to the intramolecular distances within the globular portion of the complex (see below), while the tail indicates that NTAIL possesses regions with an extended conformation.
Protein: DP04423 (UniProt P0DXN6)
Region: residues 400	to 525
Annotation: disorder (Structural state)
Evidence: sodium dodecyl sulfate polyacrylamide gel electrophoresis evidence used in manual assertion
Supporting statements:
As shown in Fig. 1B, NTAILdisplays an abnormally slow migration in SDS-PAGE even after heat denaturation. In particular, it migrates with an apparent molecular mass (MM) of 20 kDa, whereas the expected MM is 15 kDa. This abnormal behavior has already been observed in the case of the intrinsically disordered MV PNT (41) as well as in other intrinsically disordered proteins and can most likely be ascribed to their unusual sequence composition (30).
Protein: DP04423 (UniProt P0DXN6)
Region: residues 400	to 525
Annotation: pre-molten globule (Structural state)
Evidence: dynamic light scattering assay evidence used in manual assertion
Supporting statements:
Thus, the V of NTAIL calculated by DLS is about 3 times larger than the value expected for a globular protein and half of that expected for a denatured, fully unfolded protein. Rather, these hydrodynamic properties are consistent with the hypothesis that NTAIL is a native premolten globule (31).