LiteFold/DisProt huggingface dataset:
Protein: DP03676 (UniProt D0NBE6)
Region: residues 378	to 381
Annotation: protein binding (Molecular function)
Evidence: point mutation phenotypic evidence used in manual assertion
Supporting statements:
Site-directed mutagenesis of the PexRD54 C-terminal AIM region, and ATG8CL binding to a PexRD54 AIM-based peptide array, mapped the key residues that define the PexRD54-ATG8CL interface.
First, we used alanine-scanning mutagenesis to substitute Ala at six positions in the PexRD54 AIM region, Pro-373, Asp-377, Trp-378, Glu-379, Ile-380, and Val-381. Each of these proteins was expressed and purified as described for wild type. We then used analytical gel filtration to qualitatively assay whether these variants support complex formation with ATG8CL. As predicted, we did not observe interaction of PexRD54 W378A with ATG8CL (Fig. 7). For each of the other mutations, we still observed an interaction with ATG8CL, including PexRD54 V381A.
FIGURE 4. Crystal structure of ATG8CL bound to the PexRD54(377–381)-peptide and specificity of peptide binding. A, schematic representation of ATG8CL/PexRD54(377–381)-peptide complex highlighting key interactions. ATG8CL is shown in magenta schematic representation with the molecular surface that contacts the PexRD54(377–381)-peptide shown in orange. The PexRD54(377–381)-peptide is shown as sticks with yellow carbon atoms. The electron density omit map of the peptide ligand (Fobs − Fcalc map) is shown in blue mesh and contoured at 2 σ. Electrostatic interactions are indicated with black dashed lines. B, results of the peptide array analyzing the effect of single amino acid substitutions (top) at all positions of 10-mer peptide of PexRD54 (Lys-372–Val-381, side). GST-tagged ATG8CL was visualized using an anti-GST-HRP antibody.
FIGURE 7. Analysis of the interaction between PexRD54 variants and ATG8CL by gel filtration. Analytical gel filtration traces were obtained for PexRD54 variants mutated in the AIM region and incubated with ATG8CL (1:1 mixture). 
Full-length RFP-PexRD54 and the AIM motif disrupting variant RFP-PexRD54378-AEIA-381 were used as controls.
The functional LIR motif WEIV in positions 378-381 is inside the disordered fragment region in positions 370-381.
Protein: DP03676 (UniProt D0NBE6)
Region: residues 378	to 381
Annotation: selective autophagy (Biological process)
Evidence: point mutation phenotypic evidence used in manual assertion
Supporting statements:
Site-directed mutagenesis of the PexRD54 C-terminal AIM region, and ATG8CL binding to a PexRD54 AIM-based peptide array, mapped the key residues that define the PexRD54-ATG8CL interface.
First, we used alanine-scanning mutagenesis to substitute Ala at six positions in the PexRD54 AIM region, Pro-373, Asp-377, Trp-378, Glu-379, Ile-380, and Val-381. Each of these proteins was expressed and purified as described for wild type. We then used analytical gel filtration to qualitatively assay whether these variants support complex formation with ATG8CL. As predicted, we did not observe interaction of PexRD54 W378A with ATG8CL (Fig. 7). For each of the other mutations, we still observed an interaction with ATG8CL, including PexRD54 V381A.
FIGURE 4. Crystal structure of ATG8CL bound to the PexRD54(377–381)-peptide and specificity of peptide binding. A, schematic representation of ATG8CL/PexRD54(377–381)-peptide complex highlighting key interactions. ATG8CL is shown in magenta schematic representation with the molecular surface that contacts the PexRD54(377–381)-peptide shown in orange. The PexRD54(377–381)-peptide is shown as sticks with yellow carbon atoms. The electron density omit map of the peptide ligand (Fobs − Fcalc map) is shown in blue mesh and contoured at 2 σ. Electrostatic interactions are indicated with black dashed lines. B, results of the peptide array analyzing the effect of single amino acid substitutions (top) at all positions of 10-mer peptide of PexRD54 (Lys-372–Val-381, side). GST-tagged ATG8CL was visualized using an anti-GST-HRP antibody.
FIGURE 7. Analysis of the interaction between PexRD54 variants and ATG8CL by gel filtration. Analytical gel filtration traces were obtained for PexRD54 variants mutated in the AIM region and incubated with ATG8CL (1:1 mixture). 
Full-length RFP-PexRD54 and the AIM motif disrupting variant RFP-PexRD54378-AEIA-381 were used as controls.
The functional LIR motif WEIV in positions 378-381 is inside the disordered fragment region in positions 370-381.
Protein: DP03676 (UniProt D0NBE6)
Region: residues 378	to 381
Annotation: selective autophagy (Biological process)
Evidence: peptide array evidence used in manual assertion
Supporting statements:
Site-directed mutagenesis of the PexRD54 C-terminal AIM region, and ATG8CL binding to a PexRD54 AIM-based peptide array, mapped the key residues that define the PexRD54-ATG8CL interface.
FIGURE 4. Crystal structure of ATG8CL bound to the PexRD54(377–381)-peptide and specificity of peptide binding. A, schematic representation of ATG8CL/PexRD54(377–381)-peptide complex highlighting key interactions. ATG8CL is shown in magenta schematic representation with the molecular surface that contacts the PexRD54(377–381)-peptide shown in orange. The PexRD54(377–381)-peptide is shown as sticks with yellow carbon atoms. The electron density omit map of the peptide ligand (Fobs − Fcalc map) is shown in blue mesh and contoured at 2 σ. Electrostatic interactions are indicated with black dashed lines. B, results of the peptide array analyzing the effect of single amino acid substitutions (top) at all positions of 10-mer peptide of PexRD54 (Lys-372–Val-381, side). GST-tagged ATG8CL was visualized using an anti-GST-HRP antibody.
The results of the peptide array clearly highlight the importance of the hydrophobic residues 378 and 381 of the PexRD54 AIM motif (Trp and Val) in binding ATG8CL. For position 378, the strongest binding was seen for Trp and Phe, with limited binding of Tyr and the aliphatic amino acids. Position 381 favors the bulky aliphatic amino acids, with limited binding also observed by bulky hydrophobic residues. Interestingly, with the exception of Pro at position 379, any amino acid can be accommodated at positions 379 and 380, and binding is still observed. Furthermore, any amino acid can be accommodated at positions 372–377 without a significant reduction in binding, suggesting that these residues may only act as a linker between the WY domain region of PexRD54 and the C-terminal AIM motif.
The functional LIR motif WEIV in positions 378-381 is inside the disordered fragment region in positions 370-381.
Protein: DP03676 (UniProt D0NBE6)
Region: residues 378	to 381
Annotation: protein binding (Molecular function)
Evidence: peptide array evidence used in manual assertion
Supporting statements:
Site-directed mutagenesis of the PexRD54 C-terminal AIM region, and ATG8CL binding to a PexRD54 AIM-based peptide array, mapped the key residues that define the PexRD54-ATG8CL interface.
FIGURE 4. Crystal structure of ATG8CL bound to the PexRD54(377–381)-peptide and specificity of peptide binding. A, schematic representation of ATG8CL/PexRD54(377–381)-peptide complex highlighting key interactions. ATG8CL is shown in magenta schematic representation with the molecular surface that contacts the PexRD54(377–381)-peptide shown in orange. The PexRD54(377–381)-peptide is shown as sticks with yellow carbon atoms. The electron density omit map of the peptide ligand (Fobs − Fcalc map) is shown in blue mesh and contoured at 2 σ. Electrostatic interactions are indicated with black dashed lines. B, results of the peptide array analyzing the effect of single amino acid substitutions (top) at all positions of 10-mer peptide of PexRD54 (Lys-372–Val-381, side). GST-tagged ATG8CL was visualized using an anti-GST-HRP antibody.
The results of the peptide array clearly highlight the importance of the hydrophobic residues 378 and 381 of the PexRD54 AIM motif (Trp and Val) in binding ATG8CL. For position 378, the strongest binding was seen for Trp and Phe, with limited binding of Tyr and the aliphatic amino acids. Position 381 favors the bulky aliphatic amino acids, with limited binding also observed by bulky hydrophobic residues. Interestingly, with the exception of Pro at position 379, any amino acid can be accommodated at positions 379 and 380, and binding is still observed. Furthermore, any amino acid can be accommodated at positions 372–377 without a significant reduction in binding, suggesting that these residues may only act as a linker between the WY domain region of PexRD54 and the C-terminal AIM motif.
The functional LIR motif WEIV in positions 378-381 is inside the disordered fragment region in positions 370-381.
Protein: DP03676 (UniProt D0NBE6)
Region: residues 378	to 381
Annotation: protein binding (Molecular function)
Evidence: surface plasmon resonance evidence used in manual assertion
Supporting statements:
FIGURE 1. Interaction of PexRD54 and ATG8CL proteins in vitro. A, analytical gel filtration traces obtained for PexRD54 (top), ATG8CL (middle), and a 1:1 mixture of the complex (bottom). Insets show SDS-polyacrylamide gels of the fractions collected across the elution peaks. B, gel filtration trace derived from preparative purification of the PexRD54-ATG8CL complex following co-expression in E. coli. Inset, SDS-polyacrylamide gel containing purified complex. C, binding curve derived from SPR single cycle kinetics data for PexRD54 binding to ATG8CL.
Finally, we used surface plasmon resonance (SPR) to investigate the affinities of complex formation between PexRD54 and ATG8CL (Fig. 1C). Using this technique, we determined that PexRD54 binds to ATG8CL with a Kd of 388 ± 47 nm. The AIM motif disrupting PexRD54378-AEIA-381 variant (where the Trp and Val of the “WEIV” AIM motif are replaced by alanine) did not bind to ATG8CL using SPR, consistent with previous results (35).
The functional LIR motif WEIV in positions 378-381 is inside the disordered fragment region in positions 370-381.
Protein: DP03676 (UniProt D0NBE6)
Region: residues 378	to 381
Annotation: selective autophagy (Biological process)
Evidence: surface plasmon resonance evidence used in manual assertion
Supporting statements:
FIGURE 1. Interaction of PexRD54 and ATG8CL proteins in vitro. A, analytical gel filtration traces obtained for PexRD54 (top), ATG8CL (middle), and a 1:1 mixture of the complex (bottom). Insets show SDS-polyacrylamide gels of the fractions collected across the elution peaks. B, gel filtration trace derived from preparative purification of the PexRD54-ATG8CL complex following co-expression in E. coli. Inset, SDS-polyacrylamide gel containing purified complex. C, binding curve derived from SPR single cycle kinetics data for PexRD54 binding to ATG8CL.
Finally, we used surface plasmon resonance (SPR) to investigate the affinities of complex formation between PexRD54 and ATG8CL (Fig. 1C). Using this technique, we determined that PexRD54 binds to ATG8CL with a Kd of 388 ± 47 nm. The AIM motif disrupting PexRD54378-AEIA-381 variant (where the Trp and Val of the “WEIV” AIM motif are replaced by alanine) did not bind to ATG8CL using SPR, consistent with previous results (35).
The functional LIR motif WEIV in positions 378-381 is inside the disordered fragment region in positions 370-381.
Protein: DP03676 (UniProt D0NBE6)
Region: residues 378	to 381
Annotation: selective autophagy (Biological process)
Evidence: circular dichroism evidence used in manual assertion
Supporting statements:
The overall fold of the PexRD54378-AEIA-381 variant was equivalent to wild-type protein as assessed by circular dichroism (CD) spectroscopy (Fig. 2).
FIGURE 2.CD spectra of PexRD54. Far-UV CD spectra of wild-type PexRD54 (solid line) and its variant PexRD54378-AEIA-381 (dashed line) confirming similar secondary structure content (predominantly α-helical).
The functional LIR motif WEIV in positions 378-381 is inside the disordered fragment region in positions 370-381.
Protein: DP03677 (UniProt P35193)
Region: residues 124	to 235
Annotation: disorder (Structural state)
Evidence: in vitro cleavage assay evidence used in manual assertion
Supporting statements:
Stable domains in Atg19 were identified by limited proteolysis of the full-length Atg19 (supplemental Fig. S1), which showed that Atg19 comprises at least two structurally stable domains, the N-terminal domain (residues 1–123) and the C-terminal domain (residues 254–367)
Protein: DP03677 (UniProt P35193)
Region: residues 124	to 235
Annotation: protein binding (Molecular function)
Evidence: glutathione S-transferase pull-down assay evidence used in manual assertion
Supporting statements:
An additional in vitro pulldown assay showed that the coiled coil domain, but not the other two domains, of Atg19 is responsible for binding with the propeptide of prApe1 (Fig. 1B).
Protein: DP03677 (UniProt P35193)
Region: residues 412	to 415
Annotation: autophagy (Biological process)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
We then went on to determine the effect of mutations in the canonical 412WEEL415 LIR on the extent and position of the CSCs in Atg8. To this end, Trp412 was mutated to Ala, and the mutant Atg19 CTD was titrated into labeled Atg8. Consistent with previous data showing that the 412WEEL415 is not essential for Atg8 interaction (24), addition of this mutant CTD still resulted in apparent CSCs (Fig. 3, A and B). Interestingly, the shifts observed for the W412A mutant were in similar positions compared with the wild-type CTD, although Tyr49 of β2 (Lys48-Pro52) was more affected by the W412A mutant Atg19 CTD. This residue is located in the L site and thus may be contacted by the amino acid at position 4 of a LIR motif. It has previously been shown to be involved in receptor binding and autophagic function (26).
Cross-references: LIRcentral:P35193
Protein: DP03677 (UniProt P35193)
Region: residues 412	to 415
Annotation: protein binding (Molecular function)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
We then went on to determine the effect of mutations in the canonical 412WEEL415 LIR on the extent and position of the CSCs in Atg8. To this end, Trp412 was mutated to Ala, and the mutant Atg19 CTD was titrated into labeled Atg8. Consistent with previous data showing that the 412WEEL415 is not essential for Atg8 interaction (24), addition of this mutant CTD still resulted in apparent CSCs (Fig. 3, A and B). Interestingly, the shifts observed for the W412A mutant were in similar positions compared with the wild-type CTD, although Tyr49 of β2 (Lys48-Pro52) was more affected by the W412A mutant Atg19 CTD. This residue is located in the L site and thus may be contacted by the amino acid at position 4 of a LIR motif. It has previously been shown to be involved in receptor binding and autophagic function (26).
Cross-references: LIRcentral:P35193
Protein: DP03677 (UniProt P35193)
Region: residues 412	to 415
Annotation: protein binding (Molecular function)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
It was known that the C-terminal six residues of Atg19 are important for the interaction with Atg8 (Shintani et al. 2002). To determine the minimum region of Atg19 required for the interaction with Atg8, we studied their interaction by NMR spectroscopy, which showed that the C-terminal 8 residues are mainly involved in the interaction (Supporting Information Fig. S3A). Furthermore, in vitro pull-down assay using Atg19 mutants showed that both Trp412 and Leu415 residues are required for the interaction with Atg8 (Fig. 2C). In order to further elucidate the interaction between Atg8 and Atg19, we determined the crystal structure of Atg8 complexed with a tetra peptide, WEEL (Fig. 2A, Supporting Information Table S2, Supporting Information Fig. S4).
Cross-references: LIRcentral:P35193
Protein: DP03677 (UniProt P35193)
Region: residues 412	to 415
Annotation: autophagy (Biological process)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
It was known that the C-terminal six residues of Atg19 are important for the interaction with Atg8 (Shintani et al. 2002). To determine the minimum region of Atg19 required for the interaction with Atg8, we studied their interaction by NMR spectroscopy, which showed that the C-terminal 8 residues are mainly involved in the interaction (Supporting Information Fig. S3A). Furthermore, in vitro pull-down assay using Atg19 mutants showed that both Trp412 and Leu415 residues are required for the interaction with Atg8 (Fig. 2C). In order to further elucidate the interaction between Atg8 and Atg19, we determined the crystal structure of Atg8 complexed with a tetra peptide, WEEL (Fig. 2A, Supporting Information Table S2, Supporting Information Fig. S4).
Cross-references: LIRcentral:P35193
Protein: DP03677 (UniProt P35193)
Region: residues 364	to 415
Annotation: disorder (Structural state)
Evidence: curator inference based on intrinsic disorder prediction
Supporting statements:
Region 364 - 415 is predicted as intrinsically disordered in MobiDB (https://mobidb.org/P35193), as per Annotation source: AlphaFold-disorder.
Protein: DP03680 (UniProt P49023)
Region: residues 1	to 20
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
CD spectra of LD1, LD3 and LD5 peptides showed characteristics of random coil (Fig 2a, S3 Table) thus validating that the LD1, LD3 and LD5 motifs could exist as unfolded effector binding sites (not available for interaction) in our study and could fold upon undergoing allosteric changes after binding to their respective targets.
However, LD1, LD3, LD5, B2 and C3 do not show the characteristic peaks of secondary structures, thus may behave as unfolded effector binding sites.
LD1 region is comprised of residues 1–20.
Protein: DP03680 (UniProt P49023)
Region: residues 213	to 232
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
CD spectra of LD1, LD3 and LD5 peptides showed characteristics of random coil (Fig 2a, S3 Table) thus validating that the LD1, LD3 and LD5 motifs could exist as unfolded effector binding sites (not available for interaction) in our study and could fold upon undergoing allosteric changes after binding to their respective targets.
However, LD1, LD3, LD5, B2 and C3 do not show the characteristic peaks of secondary structures, thus may behave as unfolded effector binding sites.
LD3 region is cormprised of residues 213–232.
Protein: DP03680 (UniProt P49023)
Region: residues 296	to 315
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
CD spectra of LD1, LD3 and LD5 peptides showed characteristics of random coil (Fig 2a, S3 Table) thus validating that the LD1, LD3 and LD5 motifs could exist as unfolded effector binding sites (not available for interaction) in our study and could fold upon undergoing allosteric changes after binding to their respective targets.
However, LD1, LD3, LD5, B2 and C3 do not show the characteristic peaks of secondary structures, thus may behave as unfolded effector binding sites.
LD5 region is comprised of residues 296–315.
Protein: DP03681 (UniProt P55285)
Region: residues 592	to 607
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Lack of electron density in this region, as shown in the PDB, is typical of IDR.
Cross-references: PDB:5VEB
Protein: DP03682 (UniProt Q13043)
Region: residues 326	to 401
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
We expressed and purified the linker region between the kinase and SARAH domains of Mst1 (residues 326–401), which is also known as the inhibitory domain. By CD and one-dimensional NMR analysis (SI Fig. 8), we found that most of the residues in this region form a disordered structure, which can provide flexibility for domain motions in the Mst1 protein.
The helix and b-sheet contents of the purified linker region of Mst1 were calculated to be 20% and 3%, respectively. These CD data indicate a lack of regular secondary structure in the linker region of human Mst1.
Protein: DP03682 (UniProt Q13043)
Region: residues 326	to 401
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
We expressed and purified the linker region between the kinase and SARAH domains of Mst1 (residues 326–401), which is also known as the inhibitory domain. By CD and one-dimensional NMR analysis (SI Fig. 8), we found that most of the residues in this region form a disordered structure, which can provide flexibility for domain motions in the Mst1 protein.
All backbone amide signals appear in a narrow range near 8.0 ppm, and there are no aliphatic signals below 0.7 ppm, indicating the presence of an unfolded structure.
Protein: DP03682 (UniProt Q13043)
Region: residues 343	to 357
Annotation: protein binding (Molecular function)
Evidence: fluorescence polarization evidence used in manual assertion
Supporting statements:
MST1 T353 and T367 peptides displayed high phospho-dependent binding affinities for MOB1A, with Kds of 280 nm and 680 nm for the phosphorylated peptides, respectively, and no detectable binding for the nonphosphorylated peptides (Fig. 2A).
Protein: DP03682 (UniProt Q13043)
Region: residues 358	to 372
Annotation: protein binding (Molecular function)
Evidence: fluorescence polarization evidence used in manual assertion
Supporting statements:
MST1 T353 and T367 peptides displayed high phospho-dependent binding affinities for MOB1A, with Kds of 280 nm and 680 nm for the phosphorylated peptides, respectively, and no detectable binding for the nonphosphorylated peptides (Fig. 2A).
Protein: DP03682 (UniProt Q13043)
Region: residues 343	to 357
Annotation: protein binding (Molecular function)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
As predicted by prior mutational data, the MST1 pT353 and pT367 phosphopeptides bound to the MOB1A phospho-binding pocket first identified by Rock et al. (22), with the phosphate of the P0 threonine directly coordinated by K153, R154 and R157.
Cross-references: PDB:5TWG
PDB:5TWH
Protein: DP03683 (UniProt Q13137)
Region: residues 365	to 413
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
In the final structure, we could well define the extreme C-terminal region including the second ‘CPXC’ motif of CALCOCO2 (residues 414–446, referred hereafter as “ZF2”) that consists of 2 anti-parallel β-strands and a C-terminal α-helix, but we could only define a short α-helix in the ZF1 domain due to its intrinsic dynamic property, whereas the remaining region was too flexible to be well defined (Fig. S2C and D).
Cross-references: BMRB:25423
Protein: DP03683 (UniProt Q13137)
Region: residues 127	to 141
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Unfortunately, despite many attempts, we failed to solve the complex structure either using x-ray crystallography or NMR spectroscopy, likely due to the weak and dynamic nature of this RB1CC1 Claw and NDP52 LIR interaction.
Cross-references: PDB:7EAA