LiteFold/DisProt huggingface dataset:
Protein: DP03487 (UniProt Q9GZZ9)
Region: residues 338	to 346
Annotation: selective autophagy (Biological process)
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".
Protein: DP03487 (UniProt Q9GZZ9)
Region: residues 333	to 348
Annotation: selective autophagy (Biological process)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
An atypical LIR motif within UBA5 (ubiquitin like modifier activating enzyme 5) interacts with GABARAP proteins and mediates membrane localization of UBA5
Structural analysis of GABARAP proteins in complex with the UBA5 LIR peptide reveals a new molecular mechanism of Atg8:LIR interactions
We previously showed that UBA5 has an unusual LIR motif consisting of the core sequence EWGIELVSE, which predominantly interacts with GABARAP proteins [24] and does not align with other known LIR motifs [3]. To understand the molecular mechanism of this interaction, we solved the NMR solution structure of GABARAPL2 in complex with a peptide spanning residues 333–348 of UBA5 (Figure 1(a).).
The region includes the non-canonical LIR motif "WGIELV".
Cross-references: PDB:6H8C
Protein: DP03487 (UniProt Q9GZZ9)
Region: residues 333	to 348
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
An atypical LIR motif within UBA5 (ubiquitin like modifier activating enzyme 5) interacts with GABARAP proteins and mediates membrane localization of UBA5
Structural analysis of GABARAP proteins in complex with the UBA5 LIR peptide reveals a new molecular mechanism of Atg8:LIR interactions
We previously showed that UBA5 has an unusual LIR motif consisting of the core sequence EWGIELVSE, which predominantly interacts with GABARAP proteins [24] and does not align with other known LIR motifs [3]. To understand the molecular mechanism of this interaction, we solved the NMR solution structure of GABARAPL2 in complex with a peptide spanning residues 333–348 of UBA5 (Figure 1(a).).
The region includes the non-canonical LIR motif "WGIELV".
Cross-references: PDB:6H8C
Protein: DP03487 (UniProt Q9GZZ9)
Region: residues 333	to 348
Annotation: protein binding (Molecular function)
Evidence: peptide array evidence used in manual assertion
Supporting statements:
An atypical LIR motif within UBA5 (ubiquitin like modifier activating enzyme 5) interacts with GABARAP proteins and mediates membrane localization of UBA5
To analyze the importance of each hydrophobic residue within the core UBA5 LIR motif for the binding to GABARAP proteins, we performed peptide arrays, in which we mutated residues W341, I343, L345 and V346 of UBA5 to other hydrophobic amino acids (W, F, Y, I, L, V, M and A). In agreement with our structural analysis, mutations of I343 and L345, which occupy HP1, as well as V346, which occupies HP2, results in a moderate decrease in binding affinity of up to 30% (Figure 1(d)). Thus, these UBA5 LIR residues do not form the critical specific contacts to GABARAPs and could be substituted by any hydrophobic residues without drastic effects. In contrast, W341 appears to be crucial for binding: only aromatic amino acids are tolerated at this position and mutations to any other aliphatic residue abrogated the interaction almost completely (Figure 1(d)). This is in line with our previous results [24], where mutating W341 to alanine (W341A) resulted in a loss of binding of the full-length UBA5 to GABARAPs in pull-down experiments, while the mutations I343A, L345A and V346A only partially reduced the interaction.
The region includes the non-canonical LIR motif "WGIELV".
Cross-references: PDB:6H8C
Protein: DP03487 (UniProt Q9GZZ9)
Region: residues 333	to 348
Annotation: localization (Biological process)
Evidence: immunofluorescence confocal microscopy evidence used in manual assertion
Supporting statements:
An atypical LIR motif within UBA5 (ubiquitin like modifier activating enzyme 5) interacts with GABARAP proteins and mediates membrane localization of UBA5
The interaction between UBA5 and GABARAP proteins is crucial for UBA5 localization to ER membranes
Taken together, we could show that GABARAP proteins regulates the cellular localization of UBA5 by recruiting it to ER membranes and thus serves as a recruitment factor for UBA5. Consequently, this UBA5 localization predetermined the functional activity of the UFL1/UFBP1 E3 complexes within the ER, highlighting the role of GABARAPs as a signaling scaffold in the ufmylation pathway.
The region includes the non-canonical LIR motif "WGIELV".
Cross-references: PDB:6H8C
Protein: DP03487 (UniProt Q9GZZ9)
Region: residues 338	to 346
Annotation: protein ufmylation (Biological process)
Evidence: direct assay evidence used in manual assertion
Supporting statements:
Structural and Functional Analysis of a Novel Interaction Motif within UFM1-activating Enzyme 5 (UBA5) Required for Binding to Ubiquitin-like Proteins and Ufmylation.
Formation of UFM1 conjugates depends on intact LIR/UFIM within UBA5.
Finally, we tested whether conjugation of UFM1 to target proteins in the cell depended on the intact LIR/UFIM in UBA5. We made use of the cellular ufmylation assay in which FLAG-tagged wild-type and LIR/UFIM mutant forms of UBA5 are co-expressed with the C-terminal glycine-exposed form of FLAG-UFM1 as well as Myc-UFL1 and Myc-UFBP1 (the latter serves as a substrate for ufmylation) in HEK293 cells in which UBA5 was deleted by CRISPR/Cas9 technology (4). UFBP1 is an E3 adaptor protein, dependent on its Lys-267 ufmylation (3); therefore, we used wild-type UFBP1 and its K267R mutant form as a negative control in this experiment. Upon overexpression of wild-type FLAG-UBA5, or the W341A and G342A single mutants, we observed increased formation of ufmylated UFBP1 species (UFBP1-UFM1, Fig. 5B) and the uncharacterized UFM1-protein conjugates (X-UFM1), for which ufmylation in turn depends on ufmylated UFBP1 (Fig. 5B).
We thus concluded that the intact LIR/UFIM is the prerequisite for the full biological activity of UBA5 toward activation and transfer of UFM1 to downstream substrates both in vitro and in cells.
The region includes the non-canonical LIR motif "WGIELV".
Protein: DP03488 (UniProt P43026)
Region: residues 382	to 396
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The N-terminal residues 1–14 are not visible in the electron density maps, most likely because they are disordered in the solvent.
Region 1-15 is disordered in the crystal, which corresponds to region 382-396 of the amino acid sequence, since the natural precursor form contains a signal peptide (1-27) and a propeptide (28-381).
Cross-references: PDB:2BHK
Protein: DP03489 (UniProt P0DSM4)
Region: residues 1	to 25
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Circular dichroism spectropolarimetry indicated that the ponericins are unstructured in aqueous solution but adopt α -helical conformations in lipid mimetic environments.
CD spectra of the peptides in aqueous solution were typical of disordered protein chains, with a deep spectral minimum below 200 nm. Addition of 20% TFE or 20 mM SDS induced a radical change in the CD spectrum, with minima at 207 and 222 nm and a maximum at 193 nm, features that are characteristic of α-helical sec­ondary structure [38] (Fig. 7A–C).
Protein: DP03490 (UniProt P18075)
Region: residues 293	to 327
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The structural model includes residues 6–100 of ActRII-ECD (out of 1–102) and residues 36–139 of BMP7 (out of 1–139). The N-terminal 35 amino acids of BMP7 remain disordered, as in the unbound ligand (Griffith et al. 1996).
Region 1-35 corresponds to region 293-327 of the amino acid sequence, since the natural precursor form contains a signal peptide (1-29) and a propeptide (30-292).
Cross-references: PDB:1LX5
PDB:1LXI
Protein: DP03492 (UniProt Q8R426-2)
Region: residues 160	to 170
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
In  the  final  model,  there  are  two  regions  that  lack sufficient electron density: residues 160–170 of KChIP1 and 21–30 of Kv4.2, likely due to the high conformational flexibility of these regions.
Dots represent residues 160–170  of  KChIP1*  that  lack  electron  density.
Cross-references: PDB:1S6C
Protein: DP03494 (UniProt Q04087)
Region: residues 45	to 102
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
From sequence conservation and secondary structure predictions, it appeared that the density probably represented two N-terminal segments of Lrs4, with the remainder of the protein (approximately residues 34-102) disordered in our crystals.
This complex also forms crystals isomorphous to those of the complex with Lrs4 1-102 (data not shown), supporting the inference that Lrs4 residues 34-102 are disordered in the latter crystals and hence do not contribute to the diffraction intensities.
While Lrs4 residues 34-102 were present in the complex as crystallized, they were disordered in the electron density maps.
The region spanning 34-102 corresponds to 45-102 in UniProt.
Cross-references: PDB:3N7N
Protein: DP03494 (UniProt Q04087)
Region: residues 45	to 102
Annotation: disorder (Structural state)
Evidence: electron microscopy evidence used in manual assertion
Supporting statements:
Second, the bulk of the two Lrs4 subunits is either largely disordered in solution or flexibly linked to the rest of the complex. This conclusion is consistent with our observation that residues ∼34-102 were disordered in the crystal structure of Csm1/Lrs4 1-102.
The region spanning 34-102 corresponds to 45-102 in UniProt.
Protein: DP03495 (UniProt P63883)
Region: residues 163	to 174
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The final electron density map shows a clear density for the whole protein except for five disordered segments consisting of the first fifteen residues of the N-terminal purification tag, Asn146-Asp152, Lys163-Gln174, Lys310-Phe321 and the last nine residues at the C-terminus.
Cross-references: PDB:4BIN
Protein: DP03495 (UniProt P63883)
Region: residues 310	to 321
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The final electron density map shows a clear density for the whole protein except for five disordered segments consisting of the first fifteen residues of the N-terminal purification tag, Asn146-Asp152, Lys163-Gln174, Lys310-Phe321 and the last nine residues at the C-terminus.
Cross-references: PDB:4BIN
Protein: DP03496 (UniProt P22515)
Region: residues 774	to 796
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Based on Supplementary Table 2 there is a disordered region between residues 774-796.

Cross-references: PDB:7K5J
Protein: DP03497 (UniProt P27361)
Region: residues 1	to 23
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Invisible amino acids in the N- and C-terminal in the refined ERK1 model are shown by gray dot lines.
The first 23 amino acids are missing in all chains and are presumably disordered.
Cross-references: PDB:2ZOQ
Protein: DP03498 (UniProt P06491)
Region: residues 451	to 483
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
In both molecules, the residues 451–483, 523–539, 922–924 and 1046–1054 were disordered.
Cross-references: PDB:1NO7
Protein: DP03498 (UniProt P06491)
Region: residues 523	to 538
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
In both molecules, the residues 451–483, 523–539, 922–924 and 1046–1054 were disordered.
Boundaries of the disorder region for both molecules corresponds to 523-538.
Cross-references: PDB:1NO7
Protein: DP03499 (UniProt Q04637-8)
Region: residues 1428	to 1437
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The C-terminal region of human eIF4GI consists of two a-helical domains, 4G/C1 and 4G/C2, joined by a 13 residue linker (Figures 1B and 2).
The linker connecting domains 4G/C1 and 4G/C2 is both highly polar and apparently flexible (10/13 residues were not visible in our electron density maps).
Ten residues in the interdomain linker of 4G/C (left) were not observed in the electron density maps and are shown as a polyalanine trace (violet).
At the final stages of refinement, NCS operators were re-evaluated, and restrained NCS refinement yielded a final model (residues
1234–1566, excluding 1428–1437 from the interdomain linker, rmsd = 0.2 A˚ for 323 a-carbon pairs) with an R factor of 24.4% and an R free of 29.2% (Table 1).
Cross-references: PDB:1UG3
Protein: DP03499 (UniProt Q04637-8)
Region: residues 1428	to 1437
Annotation: flexible linker (Disorder function)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The C-terminal region of human eIF4GI consists of two a-helical domains, 4G/C1 and 4G/C2, joined by a 13 residue linker (Figures 1B and 2).
The linker connecting domains 4G/C1 and 4G/C2 is both highly polar and apparently flexible (10/13 residues were not visible in our electron density maps).
Ten residues in the interdomain linker of 4G/C (left) were not observed in the electron density maps and are shown as a polyalanine trace (violet).
At the final stages of refinement, NCS operators were re-evaluated, and restrained NCS refinement yielded a final model (residues
1234–1566, excluding 1428–1437 from the interdomain linker, rmsd = 0.2 A˚ for 323 a-carbon pairs) with an R factor of 24.4% and an R free of 29.2% (Table 1).
Cross-references: PDB:1UG3
Protein: DP03499 (UniProt Q04637-8)
Region: residues 1080	to 1234
Annotation: disorder (Structural state)
Evidence: cleavage assay evidence used in manual assertion
Supporting statements:
Sequence comparisons (Figure 1A), secondary structure predictions (data not shown), and limited proteolysis combined with mass spectrometry (data not shown) permitted identification of a protease-resistant C-terminal portion of human eIF4GI that supports binding to eIF4A and Mnk1 (data not shown). Further truncation (eIF4GI[1235–1572]) yielded a two-domain protein that gives high-quality crystals with two protomers per asymmetric unit (Experimental Procedures).
Limited proteolysis of the C-terminal region of human eIF4GI (residues 1080–1600) with a panel of specific endoproteases yielded a resistant core of approximately 40 kDa, as judged by gel electrophoresis (data not shown). Matrix-assisted laser desorption ionization mass spectrometry and N-terminal sequencing of proteolytic fragments mapped a large number of proteolytic cleavage sites to residues 1080–1234.
Protein: DP03500 (UniProt P0AFC7)
Region: residues 1	to 42
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Table 2. Residues built in the models. Subunit: nuoB; Built residues - Entire complex: 43–76, 86–179, 190–220
Residues 1-42 did not produce interpretable electron densities most probably due to protein disorder.
Cross-references: PDB:7NYR
PDB:7NYU
PDB:7NYV
EMDB:EMD-12653
EMDB:EMD-12654
EMDB:EMD-12655
Protein: DP03500 (UniProt P0AFC7)
Region: residues 180	to 189
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Table 2. Residues built in the models. Subunit: nuoB; Built residues - Entire complex: 43–76, 86–179, 190–220
Residues 180-189 did not produce interpretable electron densities most probably due to protein disorder.
Cross-references: PDB:7NYR
PDB:7NYU
PDB:7NYV
EMDB:EMD-12653
EMDB:EMD-12654
EMDB:EMD-12655
Protein: DP03501 (UniProt P0AFD1)
Region: residues 1	to 10
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Table 2. Residues built in the models. Subunit: nuoE; Built residues - Entire complex: 11–166
Residues 1-10 did not produce interpretable electron densities most probably due to protein disorder.
Cross-references: PDB:7NYR
PDB:7NYU
PDB:7NYV
PDB:7NZ1
EMDB:EMD-12653
EMDB:EMD-12654
EMDB:EMD-12655
EMDB:EMD-12661
Protein: DP03502 (UniProt P0AFD6)
Region: residues 1	to 22
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Table 2. Residues built in the models. Subunit: nuoI; Built residues - Entire complex: 23–180
Residues 1-22 did not produce interpretable electron densities most probably due to protein disorder.
Cross-references: PDB:7NYR
PDB:7NYU
PDB:7NYV
PDB:7NZ1
EMDB:EMD-12653
EMDB:EMD-12654
EMDB:EMD-12655
EMDB:EMD-12661
Protein: DP03503 (UniProt P0AFE0)
Region: residues 165	to 184
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Table 2. Residues built in the models. Subunit: NuoJ; Built residues - Entire complex: 1–164
Residues 165-184 did not produce interpretable electron densities most probably due to protein disorder.
Cross-references: PDB:7NYR
PDB:7NYU
PDB:7NYV
PDB:7NYH
EMDB:EMD-12653
EMDB:EMD-12654
EMDB:EMD-12655
EMDB:EMD-12652
Protein: DP03504 (UniProt P0AFC3)
Region: residues 1	to 14
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Table 2. Residues built in the models. Subunit: NuoA; Built residues - Entire complex: 15–38, 60–127
Residues 1-14 did not produce interpretable electron densities most probably due to protein disorder.
Cross-references: PDB:7NYR
PDB:7NYU
PDB:7NYV
PDB:7NYH
EMDB:EMD-12653
EMDB:EMD-12654
EMDB:EMD-12655
EMDB:EMD-12652
Protein: DP03504 (UniProt P0AFC3)
Region: residues 39	to 59
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Table 2. Residues built in the models. Subunit: NuoA; Built residues - Entire complex: 15–38, 60–127
Residues 39-59 did not produce interpretable electron densities most probably due to protein disorder.
Cross-references: PDB:7NYR
PDB:7NYU
PDB:7NYV
PDB:7NYH
EMDB:EMD-12653
EMDB:EMD-12654
EMDB:EMD-12655
EMDB:EMD-12652
Protein: DP03504 (UniProt P0AFC3)
Region: residues 128	to 147
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Table 2. Residues built in the models. Subunit: NuoA; Built residues - Entire complex: 15–38, 60–127
Residues 128-147 did not produce interpretable electron densities most probably due to protein disorder.
Cross-references: PDB:7NYR
PDB:7NYU
PDB:7NYV
PDB:7NYH
EMDB:EMD-12653
EMDB:EMD-12654
EMDB:EMD-12655
EMDB:EMD-12652
Protein: DP03505 (UniProt Q77M43)
Region: residues 477	to 505
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
In contrast, in the free state, the same NOEs were not observed, indicating that although not a statistical random coil (Fig. 4), the unbound N is very loosely structured.
Protein: DP03505 (UniProt Q77M43)
Region: residues 486	to 503
Annotation: disorder to order (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
In contrast, residues 486–503 were strongly perturbed by binding and showed large downfield shifts in their Cα resonances, consistent with organization of this region into a helix in the bound state.
Residues 477–484 appear likely to lack any persistent structure in both the free and bound states, whereas residues 486–503 are both perturbed and markedly rigidified on binding