LiteFold/DisProt huggingface dataset:
Protein: DP03483 (UniProt P05661)
Region: residues 628	to 641
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
For this purpose, a single, nucleotide-free, continuous polypeptide EMB heavy chain model (chain C) was prepared by removing the lever arm and ELC (chain D) and adding residues 628-644 to close the discontinuity in loop 2. 
y far, the most conformationally variable portion in both isoforms resides between amino acids 626 and 645.
Protein: DP03485 (UniProt Q9VKJ9)
Region: residues 550	to 574
Annotation: flexible linker (Disorder function)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The N-terminal 24 residues connecting to the DM14 4 domain and present in the crystallized construct are flexible and disordered in the structure.
N-terminal residues 550 to 576 were disordered in both protomers and 89.79/7.92 % of the residues are within the most favored and allowed regions of a Ramachandran plot 
Flexible linker connecting domain DM14 4 and the C2 domain.
Cross-references: PDB:6EI6
Protein: DP03485 (UniProt Q9VKJ9)
Region: residues 550	to 574
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The N-terminal 24 residues connecting to the DM14 4 domain and present in the crystallized construct are flexible and disordered in the structure.
N-terminal residues 550 to 576 were disordered in both protomers and 89.79/7.92 % of the residues are within the most favored and allowed regions of a Ramachandran plot
Cross-references: PDB:6EI6
Protein: DP03486 (UniProt P05031)
Region: residues 357	to 383
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The fragment of V322 to P348 of both subunits in the structure was highly disordered; therefore, they were not included in the final drDDC model. 
Cross-references: PDB:3K40
Protein: DP03487 (UniProt Q9GZZ9)
Region: residues 325	to 404
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
A Concerted Action of UBA5 C-Terminal Unstructured Regions Is Important for transfer of Activated UFM1 to UFC1
We explored the hypothesis that the unstructured C-terminal region of UBA5 serves as a regulatory region, controlling cellular localization of the elements of the ufmylation cascade and effective interaction between them. We found that the last 20 residues in UBA5 are pivotal for binding to UFC1 and can accelerate the transfer of UFM1 to UFC1. We solved the structure of a complex of UFC1 and a peptide spanning the last 20 residues of UBA5 by NMR spectroscopy. This structure in combination with additional NMR titration and isothermal titration calorimetry experiments revealed the mechanism of interaction and confirmed the importance of the C-terminal unstructured region in UBA5 for the ufmylation cascade.
The structures of UBA5 AD (brown), UFM1 (cyan) and UFC1 (grey)
are represented as ribbon diagrams; the UBA5 unstructured C-terminus containing regions R1 (orange), R2 (green) and R3
(violet) is shown as dashed lines. The structures were generated from PDB entry 5IAA [29].
Cross-references: PDB:7OVC
BMRB:34638
Protein: DP03487 (UniProt Q9GZZ9)
Region: residues 384	to 404
Annotation: disorder to order (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Residues 394–404 of the R3 region form the predicted [32] α-helix, residues 384–392 are in an extended conformation, well-defined and occupy a specific area on the UFC1 surface. Residues 381–383 seem disordered and do not interact specifically with any UFC1 residues.
Cross-references: PDB:7OVC
BMRB:34638
Protein: DP03487 (UniProt Q9GZZ9)
Region: residues 384	to 404
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Residues 394–404 of the R3 region form the predicted [32] α-helix, residues 384–392 are in an extended conformation, well-defined and occupy a specific area on the UFC1 surface. Residues 381–383 seem disordered and do not interact specifically with any UFC1 residues.
Cross-references: PDB:7OVC
BMRB:34638
Protein: DP03487 (UniProt Q9GZZ9)
Region: residues 325	to 357
Annotation: protein binding (Molecular function)
Evidence: isothermal titration calorimetry evidence used in manual assertion
Supporting statements:
The affinity of UFM1 to the R1-containing peptides (R1-R2-R3 325–404 and R1-R2 325–376, Supplementary Figure S2B and Table 2) does not change significantly compared to the
affinity of the isolated R1 325 − 357 peptide [31], indicating that this interaction is completely located within the LIR/UFIM containing region.
While UFM1 seems to bind only to the LIR/UFIM region of UBA5, LC3/GABARAP proteins interact with additional residues outside of the of the R1 sequence.
In general, we were able to observe relatively stable interactions between members of the ufmylation cascade only for the R1:UFM1 and R3:UFC1 interactions.
Protein: DP03487 (UniProt Q9GZZ9)
Region: residues 325	to 376
Annotation: protein binding (Molecular function)
Evidence: isothermal titration calorimetry evidence used in manual assertion
Supporting statements:
In contrast, LC3/GABARAP proteins showed a 10-fold higher affinity to the R1-R2-R3 325–404 and R1-R2 325–376 peptides compared to the isolated LIR/UFIM motif (R1 337 − 348 ) characterized in [31,33]. The K D values for interactions between R1-R2-R3 325–404 and GABARAPL2 (0.17 µM) or LC3B (3.7 µM) indicate the same subfamily-specific preferences that were reported previously (Supplementary Figure S2C,D).
While UFM1 seems to bind only to the LIR/UFIM region of UBA5, LC3/GABARAP proteins interact with additional residues outside of the of the R1 sequence. LC3 and GABARAP subfamily proteins showed a 10-fold higher affinity to the complete UBA5 C-terminus compared to the isolated R1 peptide.
The GABARAP and LC3 subfamilies members were found to bind UBA5 via an atypical LIR (LIR/UFIM), an evolutionary conserved sequence within the UBA5 C-terminal part [31,33]. The ITC and NMR experiments revealed additional interactions next to the known binding site within the R1. UBA5 constructs including both R1 and R2 regions showed a 10fold higher binding affinity to all GABARAP and LC3 protein subfamily members.
Protein: DP03487 (UniProt Q9GZZ9)
Region: residues 359	to 404
Annotation: protein binding (Molecular function)
Evidence: isothermal titration calorimetry evidence used in manual assertion
Supporting statements:
The affinity of the interaction between UBA5 and UFC1 has not been characterized previously. In ITC experiments, the shortest UBA5 peptide spanning the R3 sequence (R3 388–404 ) bound to UFC1 with a K D of >11 µM. The affinity increased 3-fold for R2-R3 359–404 and R1-R2-R3 325–404 peptides (K D of 2.7 and 2.4 µM, respectively; Figure 2A and Table 2).
Taken together, we identified a UFC1-interacting region within the UBA5 C-terminus using ITC and NMR experiments. The region is slightly longer than the conserved R3 sequence which was detected previously and shows a micromolar affinity to UFC1.
Additionally, UFC1 showed interaction outside of the R3 region, binding residues within the R2 region. NMR titrations revealed that UFC1 and GABARAPL2 have a more complex binding mechanism to the UBA5 C-terminus, involving some residues in the R2 region. However, no direct interactions of all tested proteins to the isolated R2 peptide were observed.
Our ITC and NMR titration experiments revealed that the interaction between UFC1 and UBA5 is mediated mostly by the relatively short and evolutionary conserved stretch of UBA5 residues (383–404).
The complex structure in combination with the NMR and ITC titration experiments revealed that in addition to the core R3 region, residues in the region R2 contribute to the interaction. While the isolated R2 peptide does not interact with UFC1, the combination of R2 and R3 binds three times tighter than the R3 alone.
Protein: DP03487 (UniProt Q9GZZ9)
Region: residues 359	to 404
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
To understand the role of the UBA5 C-terminal region in coordination of the binding events reported above on the molecular level, we performed NMR titration experiments. In those experiments, we titrated non-labeled UFC1 and GABARAPL2 proteins to a 15 N-labeled R1-R2-R3 325–404 peptide. The NMR experiments revealed that the interaction between UFC1 and UBA5 is mediated mostly by the UBA5 residues 386–404. These residues (in contrast to the vast majority of the R1-R2-R3 325–404 resonances, which are not affected by addition of UFC1) showed a slow-to-intermediate exchange mode. The amide backbone resonances of these residues disappeared with small chemical shift perturbation (CSP) at the earlier stages of titrations and did not appear again up to an 8-fold molar excess of UFC1 (Figure 2B, the full size spectra are presented in Supplementary Figure S3D). UBA5 residues 383–386, 400 and 403 appeared to be in intermediate exchange mode (their amide backbone resonances displayed CSP with intensity change, however, they became visible at the latest titration steps). It seems, that these UBA5 residues form additional interactions with UFC1. Interestingly, a subset of the residues within the R2 region (V370, A371, Y372 and T373) displayed moderate CSPs, however, below standard deviation level (Figure 2C), possibly indicating an influence of the UBA5 A371T mutation on the recognition of UFC1.
Taken together, we identified a UFC1-interacting region within the UBA5 C-terminus using ITC and NMR experiments. The region is slightly longer than the conserved R3 sequence which was detected previously and shows a micromolar affinity to UFC1.
Additionally, UFC1 showed interaction outside of the R3 region, binding residues within the R2 region. NMR titrations revealed that UFC1 and GABARAPL2 have a more complex binding mechanism to the UBA5 C-terminus, involving some residues in the R2 region. However, no direct interactions of all tested proteins to the isolated R2 peptide were observed.
Our ITC and NMR titration experiments revealed that the interaction between UFC1 and UBA5 is mediated mostly by the relatively short and evolutionary conserved stretch of UBA5 residues (383–404).
The complex structure in combination with the NMR and ITC titration experiments revealed that in addition to the core R3 region, residues in the region R2 contribute to the interaction. While the isolated R2 peptide does not interact with UFC1, the combination of R2 and R3 binds three times tighter than the R3 alone.
Protein: DP03487 (UniProt Q9GZZ9)
Region: residues 338	to 346
Annotation: protein binding (Molecular function)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
In this study, we described and characterized a combined linear LC3-interacting region/UFM1-interacting motif (LIR/UFIM) within the C terminus of UBA5. This single motif ensures that UBA5 binds both UFM1 and light chain 3/γ-aminobutyric acid receptor-associated proteins (LC3/GABARAP), two ubiquitin (Ub)-like proteins. We demonstrated that LIR/UFIM is required for the full biological activity of UBA5 and for the effective transfer of UFM1 onto UFC1 and a downstream protein substrate both in vitro and in cells. Taken together, our study provides important structural and functional insights into the interaction between UBA5 and Ub-like modifiers, improving the understanding of the biology of the ufmylation pathway.
In the current study, we have identified a short linear motif in the C terminus of UBA5 that drives the interaction with either UFM1 or LC3/GABARAP proteins, defining this as a LIR/UFIM (for LC3-interacting region/UFM1-interacting motif). We present structural details for its interaction with UFM1 and model its interaction with GABARAPL2 and LC3B.
A key residue, Ile-343, of the LIR/UFIM occupies the hydrophobic pocket (somewhat similar to HP2 in LC3) formed by the residues of Leu-21, Val-23, Val-32, and Phe-35 of UFM1 (Fig. 4, B and C). Additionally, Trp-341 and Val-346 of UBA5 LIR/UFIM interact with Pro-28 and Val-20 of UFM1, respectively (Fig. 4B). Trp-341 does not intercalate into the hydrophobic core of UFM1 but covers a significant part of the nonpolar surface, playing an important role in the complex stabilization. Leu-345 completes the formation of the hydrophobic cluster within the complex by contacts to the hydrophobic side chains of UFM1 (Val-20, Leu-21, and Val-23) (Fig. 4B).
The region includes the non-canonical LIR motif "WGIELV".
Cross-references: PDB:5HKH
Protein: DP03487 (UniProt Q9GZZ9)
Region: residues 338	to 346
Annotation: selective autophagy (Biological process)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
In this study, we described and characterized a combined linear LC3-interacting region/UFM1-interacting motif (LIR/UFIM) within the C terminus of UBA5. This single motif ensures that UBA5 binds both UFM1 and light chain 3/γ-aminobutyric acid receptor-associated proteins (LC3/GABARAP), two ubiquitin (Ub)-like proteins. We demonstrated that LIR/UFIM is required for the full biological activity of UBA5 and for the effective transfer of UFM1 onto UFC1 and a downstream protein substrate both in vitro and in cells. Taken together, our study provides important structural and functional insights into the interaction between UBA5 and Ub-like modifiers, improving the understanding of the biology of the ufmylation pathway.
In the current study, we have identified a short linear motif in the C terminus of UBA5 that drives the interaction with either UFM1 or LC3/GABARAP proteins, defining this as a LIR/UFIM (for LC3-interacting region/UFM1-interacting motif). We present structural details for its interaction with UFM1 and model its interaction with GABARAPL2 and LC3B.
A key residue, Ile-343, of the LIR/UFIM occupies the hydrophobic pocket (somewhat similar to HP2 in LC3) formed by the residues of Leu-21, Val-23, Val-32, and Phe-35 of UFM1 (Fig. 4, B and C). Additionally, Trp-341 and Val-346 of UBA5 LIR/UFIM interact with Pro-28 and Val-20 of UFM1, respectively (Fig. 4B). Trp-341 does not intercalate into the hydrophobic core of UFM1 but covers a significant part of the nonpolar surface, playing an important role in the complex stabilization. Leu-345 completes the formation of the hydrophobic cluster within the complex by contacts to the hydrophobic side chains of UFM1 (Val-20, Leu-21, and Val-23) (Fig. 4B).
The region includes the non-canonical LIR motif "WGIELV".
Protein: DP03487 (UniProt Q9GZZ9)
Region: residues 338	to 346
Annotation: selective autophagy (Biological process)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
In this study, we described and characterized a combined linear LC3-interacting region/UFM1-interacting motif (LIR/UFIM) within the C terminus of UBA5. This single motif ensures that UBA5 binds both UFM1 and light chain 3/γ-aminobutyric acid receptor-associated proteins (LC3/GABARAP), two ubiquitin (Ub)-like proteins. We demonstrated that LIR/UFIM is required for the full biological activity of UBA5 and for the effective transfer of UFM1 onto UFC1 and a downstream protein substrate both in vitro and in cells. Taken together, our study provides important structural and functional insights into the interaction between UBA5 and Ub-like modifiers, improving the understanding of the biology of the ufmylation pathway.
In the current study, we have identified a short linear motif in the C terminus of UBA5 that drives the interaction with either UFM1 or LC3/GABARAP proteins, defining this as a LIR/UFIM (for LC3-interacting region/UFM1-interacting motif). We present structural details for its interaction with UFM1 and model its interaction with GABARAPL2 and LC3B. By combining biophysical, biochemical and cellular techniques, we have provided a detailed characterization of the new UBL-binding motif and generated evidence for its role in the ability of UBA5 to mediate UFM1 conjugation in vitro and in cells. Our data suggest that a single UBL-binding element within UBA5 is responsible for both its function as an E1 enzyme in the ufmylation pathway and the interaction with LC3/GABARAP proteins.
NMR titration experiments confirmed the ITC data for the UFM1-LIR/UFIM interaction. We observed a typical pattern for interaction of two polypeptides with a KD in the range of 10 μm (Fig. 2D). Chemical shift perturbations (CSPs) induced by titration of the nonlabeled LIR/UFIM peptide into 15N-labeled UFM1 were moderate and mostly in intermediate to slow exchange modes. In particular, CSPs of Val-20, Leu-21, Ser-22, Val-23, Ser-26, Ala-31, Val-32, Leu-33, Ala-36, Glu-39, and Ala-63 indicated strong participation of these residues in the intermolecular contacts. However, some resonances of the UFM1 residues show significant CSP in the fast exchange mode (e.g. Ser-2, Phe-6, Lys-7, Lys-19, Thr-30, Lys-34, Phe-35, Ala-37, and Phe-40) (Fig. 2D and data not shown).
The biggest CSPs are localized within the area composed of β-strand 2 and parts of α-helix 1 (residues 20–23 and 31–36), comprising a hydrophobic spot on the UFM1 surface. The UFM1 residues within long loop 1, connecting β-strands 1 and 2, remained mostly unassigned even after formation of the complex with the LIR/UFIM, in agreement with previous observations (34). However, a few assigned residues within loop 1 showed almost no CSPs, indicating a weak contribution of this element to the interaction.
The region includes the non-canonical LIR motif "WGIELV".
Protein: DP03487 (UniProt Q9GZZ9)
Region: residues 338	to 346
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
In this study, we described and characterized a combined linear LC3-interacting region/UFM1-interacting motif (LIR/UFIM) within the C terminus of UBA5. This single motif ensures that UBA5 binds both UFM1 and light chain 3/γ-aminobutyric acid receptor-associated proteins (LC3/GABARAP), two ubiquitin (Ub)-like proteins. We demonstrated that LIR/UFIM is required for the full biological activity of UBA5 and for the effective transfer of UFM1 onto UFC1 and a downstream protein substrate both in vitro and in cells. Taken together, our study provides important structural and functional insights into the interaction between UBA5 and Ub-like modifiers, improving the understanding of the biology of the ufmylation pathway.
In the current study, we have identified a short linear motif in the C terminus of UBA5 that drives the interaction with either UFM1 or LC3/GABARAP proteins, defining this as a LIR/UFIM (for LC3-interacting region/UFM1-interacting motif). We present structural details for its interaction with UFM1 and model its interaction with GABARAPL2 and LC3B. By combining biophysical, biochemical and cellular techniques, we have provided a detailed characterization of the new UBL-binding motif and generated evidence for its role in the ability of UBA5 to mediate UFM1 conjugation in vitro and in cells. Our data suggest that a single UBL-binding element within UBA5 is responsible for both its function as an E1 enzyme in the ufmylation pathway and the interaction with LC3/GABARAP proteins.
NMR titration experiments confirmed the ITC data for the UFM1-LIR/UFIM interaction. We observed a typical pattern for interaction of two polypeptides with a KD in the range of 10 μm (Fig. 2D). Chemical shift perturbations (CSPs) induced by titration of the nonlabeled LIR/UFIM peptide into 15N-labeled UFM1 were moderate and mostly in intermediate to slow exchange modes. In particular, CSPs of Val-20, Leu-21, Ser-22, Val-23, Ser-26, Ala-31, Val-32, Leu-33, Ala-36, Glu-39, and Ala-63 indicated strong participation of these residues in the intermolecular contacts. However, some resonances of the UFM1 residues show significant CSP in the fast exchange mode (e.g. Ser-2, Phe-6, Lys-7, Lys-19, Thr-30, Lys-34, Phe-35, Ala-37, and Phe-40) (Fig. 2D and data not shown).
The biggest CSPs are localized within the area composed of β-strand 2 and parts of α-helix 1 (residues 20–23 and 31–36), comprising a hydrophobic spot on the UFM1 surface. The UFM1 residues within long loop 1, connecting β-strands 1 and 2, remained mostly unassigned even after formation of the complex with the LIR/UFIM, in agreement with previous observations (34). However, a few assigned residues within loop 1 showed almost no CSPs, indicating a weak contribution of this element to the interaction.
The region includes the non-canonical LIR motif "WGIELV".
Protein: DP03487 (UniProt Q9GZZ9)
Region: residues 338	to 346
Annotation: protein binding (Molecular function)
Evidence: isothermal titration calorimetry evidence used in manual assertion
Supporting statements:
In this study, we described and characterized a combined linear LC3-interacting region/UFM1-interacting motif (LIR/UFIM) within the C terminus of UBA5. This single motif ensures that UBA5 binds both UFM1 and light chain 3/γ-aminobutyric acid receptor-associated proteins (LC3/GABARAP), two ubiquitin (Ub)-like proteins. We demonstrated that LIR/UFIM is required for the full biological activity of UBA5 and for the effective transfer of UFM1 onto UFC1 and a downstream protein substrate both in vitro and in cells. Taken together, our study provides important structural and functional insights into the interaction between UBA5 and Ub-like modifiers, improving the understanding of the biology of the ufmylation pathway.
In the current study, we have identified a short linear motif in the C terminus of UBA5 that drives the interaction with either UFM1 or LC3/GABARAP proteins, defining this as a LIR/UFIM (for LC3-interacting region/UFM1-interacting motif). We present structural details for its interaction with UFM1 and model its interaction with GABARAPL2 and LC3B. By combining biophysical, biochemical and cellular techniques, we have provided a detailed characterization of the new UBL-binding motif and generated evidence for its role in the ability of UBA5 to mediate UFM1 conjugation in vitro and in cells. Our data suggest that a single UBL-binding element within UBA5 is responsible for both its function as an E1 enzyme in the ufmylation pathway and the interaction with LC3/GABARAP proteins.
We then characterized the interaction between UBLs and a synthetic peptide spanning the human UBA5 LIR/UFIM sequence by biophysical methods. The ITC experiment using purified UFM1 protein and the LIR/UFIM peptide showed that the affinity of the UFM1-LIR/UFIM interaction (Fig. 2C, left plot, and Table 5) lies in the middle range (KD of 8 μm).
We also analyzed interactions between the LC3/GABARAP proteins and the LIR/UFIM peptide. According to the ITC data, all GABARAP-type proteins had a significant preference for LIR/UFIM binding (Fig. 3A and Table 5), with GABARAPL2 showing the highest affinity for LIR/UFIM (KD of 1.6 μm) followed by GABARAPL1 and GABARAP (with KD of 6 and 16 μm, respectively).
The region includes the non-canonical LIR motif "WGIELV".