LiteFold/DisProt huggingface dataset:
Protein: DP03683 (UniProt Q13137)
Region: residues 129	to 141
Annotation: protein binding (Molecular function)
Evidence: gel-filtration evidence used in manual assertion
Supporting statements:
Further fragment truncation analyses together with AGFC-based assays elucidated that the LIR region of NDP52 (residues 129 to 141) can specifically interact with RB1CC1 Claw domain (Fig. 3D and fig. S7D).
To further confirm our hypothesis, we designed a series of NDP52 LIR mutations including the E130A, E131A, D132A, I133S, and V136R mutations of NDP52 LIR, which are corresponding to the critical Claw-binding E6, D7, D8, I9, and L12 residues of NAP1 FIR, respectively (Fig. 4A). As expected, using AGFC- and FP-based assays, we demonstrated that those mutations of NDP52 LIR all significantly disrupt the interaction between NDP52 LIR and RB1CC1 Claw (Fig. 3C and fig. S13, B to F).
(A-F) Analytical gel filtration chromatography-based analyses of the interactions of NDP52 LIR with LC3A (A), LC3B (B), LC3C (C), GABARAP (D), GABARAPL1 (E), and GABARAPL2 (F). These results indicate that NDP52 LIR can directly bind to six different mammalian ATG8 family proteins.
Protein: DP03683 (UniProt Q13137)
Region: residues 129	to 141
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Further detailed analyses revealed that the NMR peaks corresponding to the LIR region of NDP52 in the 1H-15N HSQC spectrum of NDP52(10-141) show obvious changes when titrated with the Claw domain of RB1CC1 (Fig. 3G) but display little changes in the presence of RB1CC1(1286-1395) (Fig. 3H), further confirming that the LIR region of NDP52 is directly involved in the interaction with RB1CC1 Claw but not RB1CC1(1286-1395). Collectively, all these data clearly demonstrated that the LIR region of NDP52 contains another RB1CC1-binding site, which can specifically interact with the Claw domain of RB1CC1.
The LIR motif corresponds to de 133-136 residues.
Protein: DP03683 (UniProt Q13137)
Region: residues 135	to 139
Annotation: phosphorylation display site (Disorder function)
Evidence: in vitro protein kinase assay evidence used in manual assertion
Supporting statements:
Using in vitro phosphorylation assays, we confirmed that TBK1 can directly phosphorylate NDP52 (fig. S26). Our mass spectrometry (MS) analysis revealed that the conserved T137 residue within the NDP52 LIR region can be readily phosphorylated by TBK1 (Fig. 8A).
Together, all the above biochemical and structural results clearly demonstrated that TBK1-mediated phosphorylation of T137 in the LIR region of NDP52 can regulate the interactions of NDP52 with ATG8 family proteins.
Protein: DP03683 (UniProt Q13137)
Region: residues 128	to 140
Annotation: protein binding (Molecular function)
Evidence: fluorescence polarization evidence used in manual assertion
Supporting statements:
Consistent with our structure and sequence analyses, further quantitative FP assays uncovered that the synthetic T137-phosphorylated NDP52 LIR peptide (“ENEEDILVVpTTQG,” referred to as phos-NDP52 LIR) displays a much stronger binding ability toward ATG8 family protein than that of the unphosphorylated NDP52 LIR peptide (Fig. 8B and fig. S23).
Notably, further FP-based assays revealed that the T137-phosphorylated and unphosphorylated NDP52 LIR peptides display similar binding affinities to the RB1CC1 Claw domain (fig. S27D), and the T137-phosphorylated NDP52 LIR peptide has a much higher binding affinity to LC3C than RB1CC1 (Fig. 8B and fig. S27D).
Supplementary material shows increasing binding affinities with  LC3A, LC3B, GABARAP, GABARAPL1, and GABARAPL2, when residue T137 is phosphorylated.
Protein: DP03683 (UniProt Q13137)
Region: residues 127	to 141
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Since the NDP52 SKICH domain had been well assigned in the 1H-15N HSQC spectrum of NDP52(10-126) in our previous study (36), therefore, we could easily differentiate the NMR peaks corresponding to the LIR region from that of the SKICH domain in the 1H-15N HSQC spectrum of NDP52(10-141) by comparing the two 1H-15N HSQC spectra of NDP52(10-141) and NDP52(10-126) (fig. S8).
Figure S8 indicates that 127-141 is a disorder region.
Protein: DP03683 (UniProt Q13137)
Region: residues 130	to 140
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Addition of unlabeled LC3C to 15N-labeled NDP52 (both at 150 μM) resulted in intensity attenuation of several resonances in the C-terminal region of NDP52, i.e., residues 130–140 (Figure 3B), indicating specific interaction of LC3C with this region, consistent with domain mapping data (Figure 3A). Quantification of signal intensities revealed that three residues, Leu134, Val135, and Val136, were attenuated most severely, suggesting they directly contribute to LC3C binding (Figure 3C).
Protein: DP03683 (UniProt Q13137)
Region: residues 131	to 137
Annotation: protein binding (Molecular function)
Evidence: luminescence-based mammalian interactome mapping assay evidence used in manual assertion
Supporting statements:
To identify residues essential for the binding of LC3C in vitro, amino acids 132–137 of NDP52 were mutated individually to Ala or Ser in the context of full-length protein. Mutations of Leu134, Val135, or Val136, but not of Ile133 or other surrounding residues abrogated the interaction of NDP52 with LC3C in a LUMIER assay (Figure 3D).
Protein: DP03683 (UniProt Q13137)
Region: residues 131	to 137
Annotation: protein binding (Molecular function)
Evidence: small interfering RNA knockdown evidence used in manual assertion
Supporting statements:
To test the importance of the NDP52-LC3C interaction in vivo, we complemented cells lacking NDP52 with NDP52 alleles that differ in their ability to bind LC3C. Recruitment of LC3C and LC3B to S. Typhimurium was re-established upon complementation with wild-type NDP52 but not with NDP52 V136S, which demonstrates the crucial importance of the NDP52-LC3C interaction for antibacterial autophagy (Figure 3E).
Protein: DP03683 (UniProt Q13137)
Region: residues 131	to 137
Annotation: xenophagy (Biological process)
Evidence: small interfering RNA knockdown evidence used in manual assertion
Supporting statements:
To test the importance of the NDP52-LC3C interaction in vivo, we complemented cells lacking NDP52 with NDP52 alleles that differ in their ability to bind LC3C. Recruitment of LC3C and LC3B to S. Typhimurium was re-established upon complementation with wild-type NDP52 but not with NDP52 V136S, which demonstrates the crucial importance of the NDP52-LC3C interaction for antibacterial autophagy (Figure 3E).
Protein: DP03683 (UniProt Q13137)
Region: residues 133	to 137
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
These data, in combination with our NMR measurements, define the tripeptide Leu-Val-Val in the linker region between SKICH and coiled-coil domain as the essential CLIR motif for binding to LC3C. This tripeptide is predicted to form a β strand, like the canonical Trp-Xaa-Xaa-Leu LIR motif, but lacks the aromatic residue found in canonical LIR motifs (Figure 3F).
Protein: DP03683 (UniProt Q13137)
Region: residues 128	to 141
Annotation: protein binding (Molecular function)
Evidence: fluorescence anisotropy evidence used in manual assertion
Supporting statements:
The CLIR peptide bound LC3C with a KD of 1.6 μM, while LC3A was bound with ∼10x lower affinity (15.1 μM) (Figures 5B and 5C).
Protein: DP03683 (UniProt Q13137)
Region: residues 128	to 141
Annotation: protein binding (Molecular function)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
LC3C bound CLIRI133W with 14-fold higher affinity than the CLIRWT peptide, resulting in the highest affinity LC3 interaction reported so far (KD = 110 nM) (Figure 5B). Importantly, the CLIRI133W peptide also bound LC3A with high affinity (290 nM), which represents a > 50-fold improvement over CLIRWT (Figure 5C). However, the specificity of NDP52 for LC3C over LC3A, defined as the ratio of KD values, diminished from 10-fold to 3-fold (Figure 5C).
Protein: DP03683 (UniProt Q13137)
Region: residues 128	to 141
Annotation: protein binding (Molecular function)
Evidence: luminescence-based mammalian interactome mapping assay evidence used in manual assertion
Supporting statements:
In contrast to the preferential interaction of wild-type NDP52 with LC3C, we observed strong and nonselective binding of NDP52I133W to all human ATG8 orthologs (Figure 5D). These data show that the NDP52 CLIR motif comprising a Leu-Val-Val sequence is able to distinguish efficiently between LC3C and its paralogs, and that mutation of the CLIR to utilize the aromatic pocket (CLIRI133W) enhances affinity but reduces the specificity for LC3C.
Protein: DP03683 (UniProt Q13137)
Region: residues 134	to 136
Annotation: protein binding (Molecular function)
Evidence: luminescence-based mammalian interactome mapping assay evidence used in manual assertion
Supporting statements:
LC3C, Bound Selectively by a Noncanonical LIR Motif in NDP52, Is Required for Antibacterial Autophagy
To identify residues essential for the binding of LC3C in vitro, amino acids 132–137 of NDP52 were mutated individually to Ala or Ser in the context of full-length protein. Mutations of Leu134, Val135, or Val136, but not of Ile133 or other surrounding residues abrogated the interaction of NDP52 with LC3C in a LUMIER assay (Figure 3D).
Region corresponding to the non canonical LIR motif "LVV" at position 134-136
Protein: DP03683 (UniProt Q13137)
Region: residues 134	to 136
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
LC3C, Bound Selectively by a Noncanonical LIR Motif in NDP52, Is Required for Antibacterial Autophagy
Addition of unlabeled LC3C to 15N-labeled NDP52 (both at 150 μM) resulted in intensity attenuation of several resonances in the C-terminal region of NDP52, i.e., residues 130–140 (Figure 3B), indicating specific interaction of LC3C with this region, consistent with domain mapping data (Figure 3A). Quantification of signal intensities revealed that three residues, Leu134, Val135, and Val136, were attenuated most severely, suggesting they directly contribute to LC3C binding (Figure 3C).
Region corresponding to the non canonical LIR motif "LVV" at position 134-136
Protein: DP03684 (UniProt P40458)
Region: residues 200	to 215
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance evidence used in manual assertion
Supporting statements:
The first 16 residues of Atg32[200–341] appeared disordered in the structure.
Cross-references: PDB:5WLP
Protein: DP03685 (UniProt P38428)
Region: residues 45	to 65
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance evidence used in manual assertion
Supporting statements:
The PDB structure from NMR indicates that the region 45-65 is disordered.
Cross-references: PDB:2MYX
Protein: DP03686 (UniProt Q5ZUV9)
Region: residues 442	to 458
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The N- and C-terminal-most residues and four flexible regions (residues 92–95, 249–253, 278–287, and 347–356) of RavZcrystal lacked defined electron density and were not modeled. 
Notice that the crystallization construct RavZ crystal used comprises residues 10–458. It lacks sequences predicted to be disordered, including the N and C termini (residues 1–9 and 459–502) and two loops (residues 23–43 and 430–440).
Cross-references: PDB:5CQC
Protein: DP03686 (UniProt Q5ZUV9)
Region: residues 1	to 46
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
RavZ structures of both fragments are resolved from residues 47 to 433 and 48 to 432, respectively, whereas the missing regions at the N- and C-termini (residues 1–47 and 433–502) might be flexible and disordered.
Cross-references: PDB:5MS8
PDB:5MS7
Protein: DP03686 (UniProt Q5ZUV9)
Region: residues 433	to 502
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
RavZ structures of both fragments are resolved from residues 47 to 433 and 48 to 432, respectively, whereas the missing regions at the N- and C-termini (residues 1–47 and 433–502) might be flexible and disordered.
Cross-references: PDB:5MS8
PDB:5MS7
Protein: DP03686 (UniProt Q5ZUV9)
Region: residues 10	to 21
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The N- and C-terminal-most residues and four flexible regions (residues 92–95, 249–253, 278–287, and 347–356) of RavZcrystal lacked defined electron density and were not modeled.
 The crystallization construct RavZcrystal is active in an Atg8/LC3 delipidation assay ((Choy et al., 2012), Figure 1A-B) and comprises residues 10-458. It lacks sequences predicted to be disordered, including the N- and C-termini (residues 1-9, 459-502) and two loops (residues 23-43, 430-440). 
Cross-references: PDB:5CQC
Protein: DP03686 (UniProt Q5ZUV9)
Region: residues 278	to 287
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The N- and C-terminal-most residues and four flexible regions (residues 92–95, 249–253, 278–287, and 347–356) of RavZcrystal lacked defined electron density and were not modeled.
 The crystallization construct RavZcrystal is active in an Atg8/LC3 delipidation assay ((Choy et al., 2012), Figure 1A-B) and comprises residues 10-458. It lacks sequences predicted to be disordered, including the N- and C-termini (residues 1-9, 459-502) and two loops (residues 23-43, 430-440). 
Cross-references: PDB:5CQC
Protein: DP03686 (UniProt Q5ZUV9)
Region: residues 347	to 357
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The N- and C-terminal-most residues and four flexible regions (residues 92–95, 249–253, 278–287, and 347–356) of RavZcrystal lacked defined electron density and were not modeled.
 The crystallization construct RavZcrystal is active in an Atg8/LC3 delipidation assay ((Choy et al., 2012), Figure 1A-B) and comprises residues 10-458. It lacks sequences predicted to be disordered, including the N- and C-termini (residues 1-9, 459-502) and two loops (residues 23-43, 430-440). 
Cross-references: PDB:5CQC
Protein: DP03686 (UniProt Q5ZUV9)
Region: residues 16	to 17
Annotation: negative regulation by symbiont of host autophagy (Biological process)
Evidence: surface plasmon resonance evidence used in manual assertion
Supporting statements:
The binding affinity between the N-terminal region of RavZ and LC3 was higher than that between the C-terminal region and LC3, and interestingly the single second LIR (LIR2) peptide had a similar binding affinity to LC3 as the intact N-terminal peptide containing the 2 LIRs, LIR1 and LIR2 (Fig. 1D). Although there are 2 LIR motifs in the N-terminal region, 2 molecules of LC3 cannot bind to this region simultaneously based on the SEC-MALS results, and the SRP data clearly showed that the LIR2 peptide had stronger binding affinity to LC3 than the LIR1 peptide. RavZ containing only the N-terminal LIRs, N-Cat, had similar binding affinity to LC3 as the single LIR2 peptide or N-terminal peptide, and the almost 50-fold stronger binding affinity between RavZ and LC3 might simply reflect a synergistically increased chance of the interaction.
Annotation related to the accessory LIR motif "FEEL".
Protein: DP03686 (UniProt Q5ZUV9)
Region: residues 29	to 32
Annotation: negative regulation by symbiont of host autophagy (Biological process)
Evidence: surface plasmon resonance evidence used in manual assertion
Supporting statements:
The binding affinity between the N-terminal region of RavZ and LC3 was higher than that between the C-terminal region and LC3, and interestingly the single second LIR (LIR2) peptide had a similar binding affinity to LC3 as the intact N-terminal peptide containing the 2 LIRs, LIR1 and LIR2 (Fig. 1D). Although there are 2 LIR motifs in the N-terminal region, 2 molecules of LC3 cannot bind to this region simultaneously based on the SEC-MALS results, and the SRP data clearly showed that the LIR2 peptide had stronger binding affinity to LC3 than the LIR1 peptide. RavZ containing only the N-terminal LIRs, N-Cat, had similar binding affinity to LC3 as the single LIR2 peptide or N-terminal peptide, and the almost 50-fold stronger binding affinity between RavZ and LC3 might simply reflect a synergistically increased chance of the interaction.
Annotation related to the LIR motif "FDLL".
Protein: DP03686 (UniProt Q5ZUV9)
Region: residues 29	to 32
Annotation: protein binding (Molecular function)
Evidence: surface plasmon resonance evidence used in manual assertion
Supporting statements:
The binding affinity between the N-terminal region of RavZ and LC3 was higher than that between the C-terminal region and LC3, and interestingly the single second LIR (LIR2) peptide had a similar binding affinity to LC3 as the intact N-terminal peptide containing the 2 LIRs, LIR1 and LIR2 (Fig. 1D). Although there are 2 LIR motifs in the N-terminal region, 2 molecules of LC3 cannot bind to this region simultaneously based on the SEC-MALS results, and the SRP data clearly showed that the LIR2 peptide had stronger binding affinity to LC3 than the LIR1 peptide. RavZ containing only the N-terminal LIRs, N-Cat, had similar binding affinity to LC3 as the single LIR2 peptide or N-terminal peptide, and the almost 50-fold stronger binding affinity between RavZ and LC3 might simply reflect a synergistically increased chance of the interaction.
Annotation related to the LIR motif "FDLL".
Protein: DP03686 (UniProt Q5ZUV9)
Region: residues 16	to 19
Annotation: protein binding (Molecular function)
Evidence: surface plasmon resonance evidence used in manual assertion
Supporting statements:
The binding affinity between the N-terminal region of RavZ and LC3 was higher than that between the C-terminal region and LC3, and interestingly the single second LIR (LIR2) peptide had a similar binding affinity to LC3 as the intact N-terminal peptide containing the 2 LIRs, LIR1 and LIR2 (Fig. 1D). Although there are 2 LIR motifs in the N-terminal region, 2 molecules of LC3 cannot bind to this region simultaneously based on the SEC-MALS results, and the SRP data clearly showed that the LIR2 peptide had stronger binding affinity to LC3 than the LIR1 peptide. RavZ containing only the N-terminal LIRs, N-Cat, had similar binding affinity to LC3 as the single LIR2 peptide or N-terminal peptide, and the almost 50-fold stronger binding affinity between RavZ and LC3 might simply reflect a synergistically increased chance of the interaction.
Annotation related to the accessory LIR motif "FEEL".
Protein: DP03686 (UniProt Q5ZUV9)
Region: residues 16	to 19
Annotation: negative regulation by symbiont of host autophagy (Biological process)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
The LC3-binding N- and C-terminal regions of RavZ are critical for inhibiting autophagy
We also performed the same experiments with point mutants, mutLIR1, mutLIR2 and mutLIR3, which possess a defect in LC3-binding (Fig. 3A). Based on our in vitro data, all double mutants formed a 1:1 complex with LC3 (Fig. 3B), and therefore must have a lower binding affinity compared with RavZ FL. Quantification of the fluorescence data indicated that a milder effect was shown by the point mutants compared with the deletion mutants (Fig. 4C), with different effects being observed with some of the point mutants.
In conclusion, the flexible N- and C-terminal regions in RavZ, and in particular the LIR motifs, are important for the recognition of LC3–PE conjugates and play a critical role in blocking autophagy in cells.
Annotation related to the accessory LIR motif "FEEL".
Protein: DP03686 (UniProt Q5ZUV9)
Region: residues 29	to 32
Annotation: negative regulation by symbiont of host autophagy (Biological process)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
The LC3-binding N- and C-terminal regions of RavZ are critical for inhibiting autophagy
We also performed the same experiments with point mutants, mutLIR1, mutLIR2 and mutLIR3, which possess a defect in LC3-binding (Fig. 3A). Based on our in vitro data, all double mutants formed a 1:1 complex with LC3 (Fig. 3B), and therefore must have a lower binding affinity compared with RavZ FL. Quantification of the fluorescence data indicated that a milder effect was shown by the point mutants compared with the deletion mutants (Fig. 4C), with different effects being observed with some of the point mutants.
In conclusion, the flexible N- and C-terminal regions in RavZ, and in particular the LIR motifs, are important for the recognition of LC3–PE conjugates and play a critical role in blocking autophagy in cells.
Annotation related to the LIR motif "FDLL".
Protein: DP03686 (UniProt Q5ZUV9)
Region: residues 435	to 438
Annotation: negative regulation by symbiont of host autophagy (Biological process)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
The LC3-binding N- and C-terminal regions of RavZ are critical for inhibiting autophagy
We also performed the same experiments with point mutants, mutLIR1, mutLIR2 and mutLIR3, which possess a defect in LC3-binding (Fig. 3A). Based on our in vitro data, all double mutants formed a 1:1 complex with LC3 (Fig. 3B), and therefore must have a lower binding affinity compared with RavZ FL. Quantification of the fluorescence data indicated that a milder effect was shown by the point mutants compared with the deletion mutants (Fig. 4C), with different effects being observed with some of the point mutants.
In conclusion, the flexible N- and C-terminal regions in RavZ, and in particular the LIR motifs, are important for the recognition of LC3–PE conjugates and play a critical role in blocking autophagy in cells.
Annotation related to the accessory LIR motif "FVTI".
Protein: DP03687 (UniProt O00410)
Region: residues 1	to 21
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
This first crystal form provided a preliminary model and included an N-terminal stretch of 21 amino acid residues, which was presumably unstructured and could not be traced in the electron density.
The peptide used in this study is derivated from the Isoform 3 of this protein.
Cross-references: PDB:6XU2
Protein: DP03687 (UniProt O00410)
Region: residues 316	to 329
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
This region of the Isoform 1 lacks electron density, as shown in the PDB, typical of IDR.
Cross-references: PDB:6XTE