LiteFold/DisProt huggingface dataset:
Protein: DP03212 (UniProt P0DTC9)
Region: residues 1	to 40
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The two globular domains of the protein (NTD and CTD) have been investigated while no high-resolution information is available yet for the flexible regions of the protein. We focus here on the 1-248 construct which comprises two disordered fragments (IDR1 and IDR2) in addition to the N-terminal globular domain (NTD) and report the sequence-specific assignment of the two disordered regions, a step forward towards the complete characterization of the whole protein.
In this frame, we provide here the backbone assignment of the two disordered regions flanking the NTD, the N-terminal IDR1 and the serine-rich disordered region IDR2, in the 1–248 residue construct (IDR1-NTD-IDR2).
The 2D HN spectrum recorded on the IDR1-NTD-IDR2 (1–248) construct of the SARS-CoV-2 nucleocapsid protein N is shown in Fig. 2. The 2D HN spectrum clearly shows a set of well-resolved NMR signals deriving from the globular NTD domain, as one can verify by superimposing the available sequence-specific assignment (BMRB 34511, Dinesh et al. 2020). In addition, a set of signals, with smaller dispersion and higher intensity, are observed. These are expected to originate from the flexible and disordered fragments of the protein (black contours in Fig. 2).
This region corresponds to IDR1.
Protein: DP03212 (UniProt P0DTC9)
Region: residues 181	to 248
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The two globular domains of the protein (NTD and CTD) have been investigated while no high-resolution information is available yet for the flexible regions of the protein. We focus here on the 1-248 construct which comprises two disordered fragments (IDR1 and IDR2) in addition to the N-terminal globular domain (NTD) and report the sequence-specific assignment of the two disordered regions, a step forward towards the complete characterization of the whole protein.
In this frame, we provide here the backbone assignment of the two disordered regions flanking the NTD, the N-terminal IDR1 and the serine-rich disordered region IDR2, in the 1–248 residue construct (IDR1-NTD-IDR2).
The 2D HN spectrum recorded on the IDR1-NTD-IDR2 (1–248) construct of the SARS-CoV-2 nucleocapsid protein N is shown in Fig. 2. The 2D HN spectrum clearly shows a set of well-resolved NMR signals deriving from the globular NTD domain, as one can verify by superimposing the available sequence-specific assignment (BMRB 34511, Dinesh et al. 2020). In addition, a set of signals, with smaller dispersion and higher intensity, are observed. These are expected to originate from the flexible and disordered fragments of the protein (black contours in Fig. 2).
This region corresponds to IDR2 and links the NTD and CTD domains.
Protein: DP03212 (UniProt P0DTC9)
Region: residues 181	to 248
Annotation: flexible linker (Disorder function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The two globular domains of the protein (NTD and CTD) have been investigated while no high-resolution information is available yet for the flexible regions of the protein. We focus here on the 1-248 construct which comprises two disordered fragments (IDR1 and IDR2) in addition to the N-terminal globular domain (NTD) and report the sequence-specific assignment of the two disordered regions, a step forward towards the complete characterization of the whole protein.
In this frame, we provide here the backbone assignment of the two disordered regions flanking the NTD, the N-terminal IDR1 and the serine-rich disordered region IDR2, in the 1–248 residue construct (IDR1-NTD-IDR2).
The 2D HN spectrum recorded on the IDR1-NTD-IDR2 (1–248) construct of the SARS-CoV-2 nucleocapsid protein N is shown in Fig. 2. The 2D HN spectrum clearly shows a set of well-resolved NMR signals deriving from the globular NTD domain, as one can verify by superimposing the available sequence-specific assignment (BMRB 34511, Dinesh et al. 2020). In addition, a set of signals, with smaller dispersion and higher intensity, are observed. These are expected to originate from the flexible and disordered fragments of the protein (black contours in Fig. 2).
This region corresponds to IDR2 and links the NTD and CTD domains.
Protein: DP03212 (UniProt P0DTC9)
Region: residues 1	to 68
Annotation: disorder (Structural state)
Evidence: fluorescence resonance energy transfer evidence used in manual assertion
Supporting statements:
This corresponds to values of persistence length (see SI) equal to 4.5 ± 0.4 and 4.3 ± 0.4 Å for the Gaussian and SAW distribution, respectively, which are similar to values reported for another unfolded protein under native conditions44–46,50. Overall, these results confirm the NTD is disordered, as predicted by sequence analysis.
Protein: DP03212 (UniProt P0DTC9)
Region: residues 172	to 245
Annotation: disorder (Structural state)
Evidence: fluorescence resonance energy transfer evidence used in manual assertion
Supporting statements:
We next turned to the linker (LINK FL) construct to investigate how the disordered region modulates the interaction and dynamics between the two folded domains.
This reconfiguration time is compatible with high internal friction effects, as observed for other unstructured proteins44,45, but may also account for the drag of the surrounding domains.
The RNA-binding domain (RBD) and dimerization domains are interconnected by a flexible disordered linker (LINK).
Protein: DP03212 (UniProt P0DTC9)
Region: residues 363	to 419
Annotation: disorder (Structural state)
Evidence: fluorescence resonance energy transfer evidence used in manual assertion
Supporting statements:
Finally, we again applied single-molecule FRET (Fig. 4A) and ns-FCS (Fig. 4B) to understand the conformational behavior of the CTD FL construct. Single-molecule FRET experiments again reveal a single population with a mean transfer efficiency of 0.59 ± 0.03 (Fig. 4A) and the denaturant dependence follows the expected trend for a disordered region, with a shift of the transfer efficiency toward lower values (Figs. 4C, S6 and S8), from 0.59 to 0.35.
Protein: DP03212 (UniProt P0DTC9)
Region: residues 174	to 247
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: microscopy evidence used in manual assertion
Supporting statements:
Similar to deletion of the folded CTD, deletion of the linkerIDR dramatically reduces formation of droplets and turbidity associated with LLPS in both conditions (Fig 5A–C).
In summary, these data suggest that in the presence of RNA, the NIDR, the linkerIDR, and the folded CTD form protein–protein and/or protein–RNA contacts important for the multivalent interactions stabilizing in vitro LLPS of N.
Protein: DP03212 (UniProt P0DTC9)
Region: residues 174	to 247
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: experimental evidence used in manual assertion
Supporting statements:
Similar to deletion of the folded CTD, deletion of the linkerIDR dramatically reduces formation of droplets and turbidity associated with LLPS in both conditions (Fig 5A–C).
In summary, these data suggest that in the presence of RNA, the NIDR, the linkerIDR, and the folded CTD form protein–protein and/or protein–RNA contacts important for the multivalent interactions stabilizing in vitro LLPS of N.
Protein: DP03212 (UniProt P0DTC9)
Region: residues 1	to 43
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: experimental evidence used in manual assertion
Supporting statements:
In summary, these data suggest that in the presence of RNA, the NIDR, the linkerIDR, and the folded CTD form protein–protein and/or protein–RNA contacts important for the multivalent interactions stabilizing in vitro LLPS of N.
Regarding the disordered regions, we found that deletion of the NIDR modestly enhanced droplet formation and turbidity in the absence of RNA (Fig 5A and B), while, in the presence of RNA, phase separation is strongly decreased (Fig 5A and C).
Protein: DP03212 (UniProt P0DTC9)
Region: residues 1	to 43
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: microscopy evidence used in manual assertion
Supporting statements:
In summary, these data suggest that in the presence of RNA, the NIDR, the linkerIDR, and the folded CTD form protein–protein and/or protein–RNA contacts important for the multivalent interactions stabilizing in vitro LLPS of N.
Regarding the disordered regions, we found that deletion of the NIDR modestly enhanced droplet formation and turbidity in the absence of RNA (Fig 5A and B), while, in the presence of RNA, phase separation is strongly decreased (Fig 5A and C).
Protein: DP03212 (UniProt P0DTC9)
Region: residues 182	to 197
Annotation: intracellular non-membrane-bounded organelle (Cellular component)
Evidence: fluorescence recovery after photobleaching evidence used in manual assertion
Supporting statements:
Nucleocapsid protein of SARS-CoV-2 associates with stress granules.
a. Alexa Fluor 488 labeled N(SARS-CoV-2) (green) colocalizes with the stress granule marker G3BP1 (red) in arsenite-treated digitonin-permeabilized HeLa cells.
FRAP of SG-associated N(SARS-CoV-2) suggested the presence of three N(SARS-CoV-2) populations (Fig. 2b): a very mobile with rapid fluorescence recovery, a slower diffusing component, and an immobile fraction, which does not recover its fluorescence after photobleaching (Fig. 2c). Because SGs consists of a rigid core and a dynamic shell, we attribute the different N(SARS-CoV-2) diffusion properties to the localization of N(SARS-CoV-2) to different sub-structures of SGs.
Protein: DP03212 (UniProt P0DTC9)
Region: residues 172	to 245
Annotation: flexible linker (Disorder function)
Evidence: fluorescence resonance energy transfer evidence used in manual assertion
Supporting statements:
The RNA-binding domain (RBD) and dimerization domains are interconnected by a flexible disordered linker (LINK).
Protein: DP03212 (UniProt P0DTC9)
Region: residues 1	to 44
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
N is a 419 amino acid multidomain protein, comprising two folded, RNA-binding and dimerization domains spanning residues 45-175 and 264-365 respectively. The remaining 164 amino acids are predicted to be intrinsically disordered, but there is currently no atomic resolution information describing their behaviour. Here we assign the backbone resonances of the first two intrinsically disordered domains (N1, spanning residues 1-44 and N3, spanning residues 176-263).
Cross-references: BMRB:50558
BMRB:50557
Protein: DP03212 (UniProt P0DTC9)
Region: residues 176	to 263
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
N is a 419 amino acid multidomain protein, comprising two folded, RNA-binding and dimerization domains spanning residues 45-175 and 264-365 respectively. The remaining 164 amino acids are predicted to be intrinsically disordered, but there is currently no atomic resolution information describing their behaviour. Here we assign the backbone resonances of the first two intrinsically disordered domains (N1, spanning residues 1-44 and N3, spanning residues 176-263).
Cross-references: BMRB:50557
BMRB:50558
Protein: DP03212 (UniProt P0DTC9)
Region: residues 222	to 230
Annotation: MHC class I protein binding (Molecular function)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
We then determined the crystal structure of six HLA-A*02:01-SARS-CoV-2 complexes, providing the first description of CD8+ T cell SARS-CoV-2 epitopes at an atomic level. Interestingly, three of our selected peptides have since been shown to be immunogenic (N219-227 (Isabel Schulien et al., 2021), N222-230 (Ferretti et al., 2020; Isabel Schulien et al., 2021), and N316-324 (Habel et al., 2020; Isabel Schulien et al., 2021)) in COVID-19 recovered individuals. As expected, these immunogenic peptides were able to form a stable complex with the HLA-A*02:01 molecule.
Overall, we demonstrate that not all selected peptides were able to form stable complexes with HLA-A*02:01, which was a consequence of unfavourable P2 and/or PΩ residues and an important factor for immunogenicity. In addition, we saw limited pre-existing CD8+ T cell response for these peptides in unexposed donors, whereas peptides that have subsequently been shown to be immunogenic in COVID-19 recovered patients were stable and adopted a canonical conformation in the cleft of HLA-A*02:01. Altogether, our data provide molecular insight into CD8+ T cell epitopes from SARS-CoV-2.
Cross-references: PDB:7KGQ
Protein: DP03212 (UniProt P0DTC9)
Region: residues 222	to 230
Annotation: MHC class I protein complex (Cellular component)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
We then determined the crystal structure of six HLA-A*02:01-SARS-CoV-2 complexes, providing the first description of CD8+ T cell SARS-CoV-2 epitopes at an atomic level. Interestingly, three of our selected peptides have since been shown to be immunogenic (N219-227 (Isabel Schulien et al., 2021), N222-230 (Ferretti et al., 2020; Isabel Schulien et al., 2021), and N316-324 (Habel et al., 2020; Isabel Schulien et al., 2021)) in COVID-19 recovered individuals. As expected, these immunogenic peptides were able to form a stable complex with the HLA-A*02:01 molecule.
Overall, we demonstrate that not all selected peptides were able to form stable complexes with HLA-A*02:01, which was a consequence of unfavourable P2 and/or PΩ residues and an important factor for immunogenicity. In addition, we saw limited pre-existing CD8+ T cell response for these peptides in unexposed donors, whereas peptides that have subsequently been shown to be immunogenic in COVID-19 recovered patients were stable and adopted a canonical conformation in the cleft of HLA-A*02:01. Altogether, our data provide molecular insight into CD8+ T cell epitopes from SARS-CoV-2.
Cross-references: PDB:7KGQ
Protein: DP03212 (UniProt P0DTC9)
Region: residues 222	to 230
Annotation: MHC class I protein complex (Cellular component)
Evidence: temperature-induced protein unfolding evidence used in manual assertion
Supporting statements:
Two peptides, N138-146 and N159-167, were not immunogenic in COVID-19-recovered individuals (Isabel Schulien et al., 2021), whereas three peptides, namely N219-227 (Isabel Schulien et al., 2021), N222-230 (Isabel Schulien et al., 2021), and N316-324 (Isabel Schulien et al., 2021; Habel et al., 2020), were immunogenic in COVID-19 recovered individuals, further highlighting the importance of further investigation into these epitopes.
The most stable pHLA complexes were the one with the N222-230 (Tm of 55°C) and N316-324 (Tm of 49°C) peptides, followed by the one with N219-227, N226-234, and N351-359 peptides with a Tm of ~40°C (Table 1, Figure S1). Surprisingly, the Tm was ~35°C for complexes with the NTD-derived peptides N138-146 and N159-167, an extremely low Tm value for pHLA-A*02:01 complexes (Valkenburg et al., 2016; Blaha et al., 2019; Khan et al., 2000).
The three peptides that resulted in a pHLA complex with a Tm above 40°C were recently described as immunogenic in COVID-19-recovered patients (N219-227 (Isabel Schulien et al., 2021), N222-230 (Ferretti et al., 2020; Isabel Schulien et al., 2021), and N316-324 (Habel et al., 2020; Isabel Schulien et al., 2021)), whereas the two peptides leading to a pHLA with a Tm below 40°C were described as non-immunogenic (N138-146 and N159-167 (Isabel Schulien et al., 2021)) (Table 1). This suggests that indeed pHLA complex stability may play a role in peptide immunogenicity.
Protein: DP03212 (UniProt P0DTC9)
Region: residues 219	to 227
Annotation: MHC class I protein complex (Cellular component)
Evidence: temperature-induced protein unfolding evidence used in manual assertion
Supporting statements:
Two peptides, N138-146 and N159-167, were not immunogenic in COVID-19-recovered individuals (Isabel Schulien et al., 2021), whereas three peptides, namely N219-227 (Isabel Schulien et al., 2021), N222-230 (Isabel Schulien et al., 2021), and N316-324 (Isabel Schulien et al., 2021; Habel et al., 2020), were immunogenic in COVID-19 recovered individuals, further highlighting the importance of further investigation into these epitopes.
The most stable pHLA complexes were the one with the N222-230 (Tm of 55°C) and N316-324 (Tm of 49°C) peptides, followed by the one with N219-227, N226-234, and N351-359 peptides with a Tm of ~40°C (Table 1, Figure S1). Surprisingly, the Tm was ~35°C for complexes with the NTD-derived peptides N138-146 and N159-167, an extremely low Tm value for pHLA-A*02:01 complexes (Valkenburg et al., 2016; Blaha et al., 2019; Khan et al., 2000).
The three peptides that resulted in a pHLA complex with a Tm above 40°C were recently described as immunogenic in COVID-19-recovered patients (N219-227 (Isabel Schulien et al., 2021), N222-230 (Ferretti et al., 2020; Isabel Schulien et al., 2021), and N316-324 (Habel et al., 2020; Isabel Schulien et al., 2021)), whereas the two peptides leading to a pHLA with a Tm below 40°C were described as non-immunogenic (N138-146 and N159-167 (Isabel Schulien et al., 2021)) (Table 1). This suggests that indeed pHLA complex stability may play a role in peptide immunogenicity.
Protein: DP03212 (UniProt P0DTC9)
Region: residues 219	to 227
Annotation: MHC class I protein binding (Molecular function)
Evidence: temperature-induced protein unfolding evidence used in manual assertion
Supporting statements:
Two peptides, N138-146 and N159-167, were not immunogenic in COVID-19-recovered individuals (Isabel Schulien et al., 2021), whereas three peptides, namely N219-227 (Isabel Schulien et al., 2021), N222-230 (Isabel Schulien et al., 2021), and N316-324 (Isabel Schulien et al., 2021; Habel et al., 2020), were immunogenic in COVID-19 recovered individuals, further highlighting the importance of further investigation into these epitopes.
The most stable pHLA complexes were the one with the N222-230 (Tm of 55°C) and N316-324 (Tm of 49°C) peptides, followed by the one with N219-227, N226-234, and N351-359 peptides with a Tm of ~40°C (Table 1, Figure S1). Surprisingly, the Tm was ~35°C for complexes with the NTD-derived peptides N138-146 and N159-167, an extremely low Tm value for pHLA-A*02:01 complexes (Valkenburg et al., 2016; Blaha et al., 2019; Khan et al., 2000).
The three peptides that resulted in a pHLA complex with a Tm above 40°C were recently described as immunogenic in COVID-19-recovered patients (N219-227 (Isabel Schulien et al., 2021), N222-230 (Ferretti et al., 2020; Isabel Schulien et al., 2021), and N316-324 (Habel et al., 2020; Isabel Schulien et al., 2021)), whereas the two peptides leading to a pHLA with a Tm below 40°C were described as non-immunogenic (N138-146 and N159-167 (Isabel Schulien et al., 2021)) (Table 1). This suggests that indeed pHLA complex stability may play a role in peptide immunogenicity.
Protein: DP03212 (UniProt P0DTC9)
Region: residues 222	to 230
Annotation: MHC class I protein binding (Molecular function)
Evidence: temperature-induced protein unfolding evidence used in manual assertion
Supporting statements:
Two peptides, N138-146 and N159-167, were not immunogenic in COVID-19-recovered individuals (Isabel Schulien et al., 2021), whereas three peptides, namely N219-227 (Isabel Schulien et al., 2021), N222-230 (Isabel Schulien et al., 2021), and N316-324 (Isabel Schulien et al., 2021; Habel et al., 2020), were immunogenic in COVID-19 recovered individuals, further highlighting the importance of further investigation into these epitopes.
The most stable pHLA complexes were the one with the N222-230 (Tm of 55°C) and N316-324 (Tm of 49°C) peptides, followed by the one with N219-227, N226-234, and N351-359 peptides with a Tm of ~40°C (Table 1, Figure S1). Surprisingly, the Tm was ~35°C for complexes with the NTD-derived peptides N138-146 and N159-167, an extremely low Tm value for pHLA-A*02:01 complexes (Valkenburg et al., 2016; Blaha et al., 2019; Khan et al., 2000).
The three peptides that resulted in a pHLA complex with a Tm above 40°C were recently described as immunogenic in COVID-19-recovered patients (N219-227 (Isabel Schulien et al., 2021), N222-230 (Ferretti et al., 2020; Isabel Schulien et al., 2021), and N316-324 (Habel et al., 2020; Isabel Schulien et al., 2021)), whereas the two peptides leading to a pHLA with a Tm below 40°C were described as non-immunogenic (N138-146 and N159-167 (Isabel Schulien et al., 2021)) (Table 1). This suggests that indeed pHLA complex stability may play a role in peptide immunogenicity.
Protein: DP03212 (UniProt P0DTC9)
Region: residues 222	to 230
Annotation: response to host immune response (Biological process)
Evidence: flow cytometry evidence used in manual assertion
Supporting statements:
SARS-CoV-2-specific memory CD8+ T cells exhibited functional characteristics comparable to influenza-specific CD8+ T cells and were detectable in SARS-CoV-2 convalescent individuals who were seronegative for anti-SARS-CoV-2 antibodies targeting spike (S) and nucleoprotein (N). These results define cross-reactive and induced SARS-CoV-2-specific CD8+ T cell responses as potentially important determinants of immune protection in mild SARS-CoV-2 infection.
Percentage of convalescent SARS-CoV-2 individuals with a positive response toward HLA-A- and HLA-B-restricted SARS-CoV-2 peptides and the strength of individual responses as percent IFN-γ+ of CD8+ T cells.
Based on Figure 1d, region 222-230 is an immunogenic peptide toward HLA-A-.
Protein: DP03212 (UniProt P0DTC9)
Region: residues 1	to 44
Annotation: immunoglobulin binding (Molecular function)
Evidence: isothermal titration calorimetry evidence used in manual assertion
Supporting statements:
We characterized the interactions between sdAbs and N‐protein using isothermal titration calorimetry (ITC). We first tested their binding to the FLN. Four out of six sdAbs showed clear binding. The Kd values of positive sdAb‐N2, sdAb‐N3, sdAb‐N5, and sdAb‐N6 are 1.75 µm, 4.37 µm, 3.97 µm, and 3.53 µm, respectively (Fig. 2). Next, we tested the binding of these four sdAbs with NLC protein. Only sdAb‐N2 and sdAb‐N3 showed positive results with Kd values of 2.24 µm and 1.09 µm, respectively (Fig. 3), indicating that the binding of sdAbs‐N5 and sdAb‐N6 requires the presence of the N‐arm or C‐tail of N‐protein.
Protein: DP03212 (UniProt P0DTC9)
Region: residues 362	to 419
Annotation: immunoglobulin binding (Molecular function)
Evidence: isothermal titration calorimetry evidence used in manual assertion
Supporting statements:
We characterized the interactions between sdAbs and N‐protein using isothermal titration calorimetry (ITC). We first tested their binding to the FLN. Four out of six sdAbs showed clear binding. The Kd values of positive sdAb‐N2, sdAb‐N3, sdAb‐N5, and sdAb‐N6 are 1.75 µm, 4.37 µm, 3.97 µm, and 3.53 µm, respectively (Fig. 2). Next, we tested the binding of these four sdAbs with NLC protein. Only sdAb‐N2 and sdAb‐N3 showed positive results with Kd values of 2.24 µm and 1.09 µm, respectively (Fig. 3), indicating that the binding of sdAbs‐N5 and sdAb‐N6 requires the presence of the N‐arm or C‐tail of N‐protein.
Protein: DP03212 (UniProt P0DTC9)
Region: residues 1	to 43
Annotation: protein homodimerization activity (Molecular function)
Evidence: quantitative mass spectrometry evidence used in manual assertion
Supporting statements:
Removal of the Narm and Carm (NNTD-LKR-CTD) results in two major species that are similar to NWT. However, both NNTD-LKR-CTD populations display reduced polydispersity (narrower peak width, Figure S2A), suggesting that both Narm and Carm contribute to N oligomerization.