A proteome-wide biochemical screen defines binding determinants of the core autophagy protein LC3B

  1. Jennifer Kosmatka
  2. Cong Liu
  3. Jackson C Halpin
  4. Daniel Lim
  5. Joseph H Davis
  6. Amy E Keating

  • Curated by eLife

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    eLife Assessment

    This study is a valuable contribution that comprehensively identifies and characterizes LC3B-binding peptides through a bacterial cell-surface display screen covering approximately 500,000 human peptides. The data presented are solid, although this approach has some limitations. For example, it cannot assess the effects of post-translational modifications, which are often relevant for LIR-mediated interactions. Validation of the newly identified binding peptides by demonstrating their interactions with full-length proteins in cells would further strengthen this work.

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Abstract

Human MAP1LC3B (LC3B) binds proteins involved in autophagy and other cellular processes using a degenerate four-residue short linear motif known as the LC3-interacting region (LIR). Biochemical and structural studies have identified LIRs in many LC3B interaction partners, but the sequence features that contribute to binding have not been systematically explored. To discover peptides that interact with LC3B and deeply profile the key binding determinants, we screened a library of ∼500,000 36-residue peptides derived from the human proteome using bacterial cell-surface display. Analysis of the screening data, coupled with structural studies and site-directed mutagenesis, revealed exceptions to the canonical LIR motif ([FWY]0-X1-X2-[LVI]3) and a strong preference for negatively charged residues adjacent to the LIR, which we visualized in a newly determined structure of LC3B bound to a peptide isolated in our screen. Guided by our screening data, we designed synthetic LIR-containing peptides that bind LC3B with affinities comparable to the tightest measured natural binder. Finally, we determined that mutations in LC3B commonly thought to abrogate binding of LIR-containing peptides instead alter LC3B binding specificity, leading to enhanced binding of some LIR-containing sequences. Taken together, our results refine the LIR motif definition, expand the network of candidate LC3B interaction partners, and highlight how mutations at the LIR docking site can modulate affinity and specificity.

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  1. Version published to 10.7554/elife.110318.2 on eLife
  2. Version published to 10.7554/elife.110318 on eLife
  3. eLife

    eLife Assessment

    This study is a valuable contribution that comprehensively identifies and characterizes LC3B-binding peptides through a bacterial cell-surface display screen covering approximately 500,000 human peptides. The data presented are solid, although this approach has some limitations. For example, it cannot assess the effects of post-translational modifications, which are often relevant for LIR-mediated interactions. Validation of the newly identified binding peptides by demonstrating their interactions with full-length proteins in cells would further strengthen this work.

  4. eLife

    Reviewer #1 (Public review):

    Summary:

    This study uses high-throughput bacterial cell surface display to identify LC3B interacting peptides in the human proteome. The screen is unbiased and this type of assay has not previously been used for selecting LC3B interacting peptides. The screen was done with library of 500,000 peptides and they ended up with 427 peptides that they scored as high-confidence LC3B binders. The experiments performed are solid and data are analyzed using well documented methods and statistics.

    The aim for the authors was to isolate LC3B interacting peptides from the human proteome, and the screen succeeded in doing so. The selected set of peptides included several previously reported LIR motifs, but also many novel LC3B binding peptides that either contained or did not contain the canonical core LIR motif [WFY]xx[LVI].

    Another aim was to identify binding determinants important for the LC3B interaction, and they made an interesting sequence logo based on selected LIR containing peptides. This study does not really extend our knowledge related to binding determinants essential for LIR motifs in LC3B binding, but it expands the number of exceptions to the canonical core LIR motif.

    Strengths:

    The approach used here (high throughput bacterial-surface-display) is new. The screen is unbiased and the fact that peptides are directly tested for LC3B binding may facilitate the discovery of non-canonical LIR motifs. The screen appears to be highly selective and manage to distinguish between peptides that interact with LC3B and peptides that do not interact.

    Weaknesses:

    It is a limitation that no proteins are analyzed in this study. Further work is therefore needed to verify that identified LIR motifs are functional in full-length proteins and in cells.

    Comments on revised version.

    It remains a weakness that none of the identified LIR motifs are tested in full-length proteins or cells, but all the specific comments in my initial review have been addressed.

  5. eLife

    Reviewer #2 (Public review):

    Summary:

    To discover peptides that interact with autophagy-related protein LC3B and profile the key binding determinants, the authors screened a library of ~500,000 36-residue peptides derived from the human proteome using bacterial cell-surface display. Analysis of the screening data revealed exceptions to the reported LIR motif and a strong preference for negatively charged residues adjacent to the LIR.
    These results support a refinement the LIR motif definition and expand the network of candidate LC3B interaction partners.

    Strengths:

    High-throughput approach

    Weaknesses:

    Lack of in vitro data and molecular dynamics simulations

    Comments on revised version.

    In the revised manuscript the authors did not provide in vitro and molecular dynamics simulations data.

  6. eLife

    Reviewer #3 (Public review):

    Summary:

    The LC3 family of proteins, which includes LC3B, are ubiquitin-like proteins that are covalently linked to phosphatidylethanolamine in the expanding autophagosomal membrane during autophagy. LC3 family members bind to short sequences of amino acids that reside within dynamic regions in a wide variety of proteins. These sequences, termed LC3 Interacting Regions (LIRs), were initially thought to function primarily to link LIR containing autophagy cargo receptors to LC3 family members to help facilitate their capture during autophagy. However, the functional importance of LIRs in autophagy has broadened to include more general functions in autophagy as well. While a general consensus for LIR sequences has been described as [FWY]0-X1-X2-[LVI]3 recent work has suggested that additional sequences outside of the canonical LIR sequence can bind LC3 family members and play important roles in autophagy. In this manuscript by Kosmatka et al, the authors perform a high throughput screen using bacterial surface display coupled with fluorescence associated cell sorting to identify which human sequences can bind to LC3B. They identify a variety of peptides capable of binding LC3B including peptides from proteins that have not previously been described as LC3B binding proteins. The results from the bacterial surface display were then used to guide sequence analysis, mutational analysis and structural studies to further characterize the range of LIR sequences that are capable of binding LC3B. Taken together, this work adds to the growing knowledge of how LIR sequences interact with LC3 family members and demonstrates which amino acids both inside and outside of the LIR sequence aid in binding. This work also identifies new potential LC3 binding proteins which may play unknown roles in autophagy regulation. Lastly, this work reinforces the importance of alternative LIR sequences such as the [WFY]0-X1-X2-[WFY]3 sequence which the authors have dubbed the LIR+ sequence.

    Strengths:

    The manuscript uses a robust approach to identify and characterize different peptide sequences that can interact with LC3B. They validate a large number of sequences using biolayer interferometry (BLI). The large number of LC3B binding sequences and their dissociation constants adds significant new information to the field that will help others understand what sequences can bind to LC3B. The authors are also very careful to accurately report on their data and not overly interpret their findings.

    Weaknesses:

    After the authors identify proteins from their bacterial display assay, the remainder of the manuscript is focused on characterizing the different types of sequences that are identified in addition to validating the LC3B-LIR interactions using BLI and X-ray crystallography. However, it's not entirely clear if the screen identified novel LC3B binders that interact with LC3B in cells. While I acknowledge that the focus of the manuscript is on the characterization of LIR sequences that can bind LC3B it seems like a missed opportunity to not validate a few of the novel LC3B binders in vivo.

  7. eLife

    Author response:

    The following is the authors’ response to the original reviews.

    Public Reviews:

    Reviewer #1 (Public review):

    Summary:

    This study uses high-throughput bacterial cell-surface display to identify LC3B-interacting peptides in the human proteome. The screen is unbiased, and this type of assay has not previously been used for selecting LC3B-interacting peptides. The screen was done with a library of 500,000 peptides, and they ended up with 427 peptides that they scored as high-confidence LC3B binders. The experiments performed are solid, and data are analyzed using well-documented methods and statistics.

    The aim of the authors was to isolate LC3B-interacting peptides from the human proteome, and the screen succeeded in doing so. The selected set of peptides included several previously reported LIR motifs, but also many novel LC3B binding peptides that either contained or did not contain the canonical core LIR motif [WFY]xx[LVI].

    Another aim was to identify binding determinants important for the LC3B interaction, and they made an interesting sequence logo based on selected LIR-containing peptides. However, this study does not really extend our knowledge related to binding determinants essential for LIR motifs in LC3B binding. They basically verify known characteristics, including the importance of varied types of electrostatic interactions supporting the docking of the core LIR into the LDS of LC3B.

    Strengths:

    The approach used here (high-throughput bacterial-surface-display) is new. The screen is unbiased, and the fact that peptides are directly tested for LC3B binding may facilitate the discovery of non-canonical LIR motifs. The screen appears to be highly selective and manages to distinguish between peptides that interact with LC3B and peptides that do not interact.

    Weaknesses:

    It is a limitation that no proteins are analyzed in this study. Further work is therefore needed to verify that identified LIR motifs are functional in full-length proteins and in cells.

    Reviewer #2 (Public review):

    Summary:

    To discover peptides that interact with autophagy-related protein LC3B and profile the key binding determinants, the authors screened a library of ~500,000 36-residue peptides derived from the human proteome using bacterial cell-surface display. Analysis of the screening data revealed exceptions to the reported LIR motif and a strong preference for negatively charged residues adjacent to the LIR.

    These results support a refinement of the LIR motif definition and expand the network of candidate LC3B interaction partners.

    Strengths:

    High-throughput approach.

    Weaknesses:

    Lack of in vitro data and molecular dynamics simulations.

    Reviewer #3 (Public review):

    Summary:

    The LC3 family of proteins, which includes LC3B, are ubiquitin-like proteins that are covalently linked to phosphatidylethanolamine in the expanding autophagosomal membrane during autophagy. LC3 family members bind to short sequences of amino acids that reside within dynamic regions in a wide variety of proteins. These sequences, termed LC3 Interacting Regions (LIRs), were initially thought to function primarily to link LIR-containing autophagy cargo receptors to LC3 family members to help facilitate their capture during autophagy. However, the functional importance of LIRs in autophagy has broadened to include more general functions in autophagy as well. While a general consensus for LIR sequences has been described as [FWY]0-X1-X2-[LVI]3, recent work has suggested that additional sequences outside of the canonical LIR sequence can bind LC3 family members and play important roles in autophagy. In this manuscript by Kosmatka et al, the authors perform a high-throughput screen using bacterial surface display coupled with fluorescence-associated cell sorting to identify which human sequences can bind to LC3B. They identify a variety of peptides capable of binding LC3B, including peptides from proteins that have not previously been described as LC3B-binding proteins. The results from the bacterial surface display were then used to guide sequence analysis, mutational analysis, and structural studies to further characterize the range of LIR sequences that are capable of binding LC3B. Taken together, this work adds to the growing knowledge of how LIR sequences interact with LC3 family members and demonstrates which amino acids both inside and outside of the LIR sequence aid in binding. This work also identifies new potential LC3 binding proteins, which may play unknown roles in autophagy regulation. Lastly, this work reinforces the importance of alternative LIR sequences such as the [WFY]0-X1-X2-[WFY]3 sequence, which the authors have dubbed the LIR+ sequence.

    Strengths:

    The manuscript uses a robust approach to identify and characterize different peptide sequences that can interact with LC3B. They validate a large number of sequences using biolayer interferometry (BLI) and attempt to correlate different amino acids with their binding affinity for LC3B. The large number of LC3B binding sequences and their dissociation constants adds significant new information to the field that will help others understand what sequences can bind to LC3B. The authors are also very careful to accurately report on their data and not overly interpret their findings.

    Weaknesses:

    After the authors identify proteins from their bacterial display assay, the remainder of the manuscript is focused on characterizing the different types of sequences that are identified in addition to validating the LC3B-LIR interactions using biochemical approaches, including BLI and X-ray crystallography. However, it's not entirely clear if the screen identified novel LC3B binders that interact with LC3B in cells. While I acknowledge that the focus of the manuscript is on the characterization of LIR sequences that can bind LC3B, it seems like a missed opportunity not to validate a few of the novel LC3B binders in vivo. This may result in the demonstration of novel binders of LC3B in cells and may further demonstrate the strength of this approach for identifying LC3 family member binding partners. Therefore, it would be helpful to look at a few proteins identified in the HC set that have not previously been identified as LC3B binders in cells to determine if they CO-IP with LC3B or interact with LC3B using a different approach.

    The reviewers appreciated the contribution of our screen and our biochemical analyses and indicated there would be additional value in performing follow-up studies in cells to investigate the functional relevance of interactions we detected. We agree that such work will be important, but it is beyond the scope of our current study. We expect the data and analyses included in this paper, and the lists of peptide binders that we identified, will inspire such future efforts.

    We appreciate the reviewers’ careful reading of our manuscript, and we have revised the text and figures to improve the clarity and presentation of the data in accordance with their recommendations.

    Recommendations for the authors:

    Reviewer #1 (Recommendations for the authors):

    Main comments:

    (1) In the abstract, the authors correctly conclude in the final sentence: "our results refine the LIR motif definition and expand the network of candidate LC3B interaction partners", but I disagree that their study "highlights how mutations at the LC3B-LIR interface can modulate affinity and specificity". They probably refer to the data in Figure 5, where they found that some peptides were positively affected by a mutation of the LDS in LC3B. This is interesting, but four peptides are listed in Figure 5B, and for these peptides, no experimental data are included to verify that their interactions with LC3B are LIR-LDS interactions. LIR-mutated peptides are, for instance, not tested. Therefore, it is possible that these interactions are not LIR-LDS interactions but depend on another binding surface in LC3B than LDS.

    Although LIR-mutated peptides were not tested in the experiments reported in Figure 5B, the LIR docking site (LDS) mutations F52A/L53A change the peptide-LC3B affinity. This suggests that the LDS is involved in the binding interaction, even if the interaction is not with a LIR motif. We have changed the statement to “highlight how mutations at the LIR docking site can modulate affinity and specificity,” to be more explicit.

    (2) Another text line in the abstract is that their study "revealed exceptions to the reported LIR motif and a strong preference for negatively charged residues adjacent to the LIR", and I find this text line a bit misleading. Yes, their structural data for the BLM peptide nicely shows the importance of electrostatic interactions, and R70 in LC3B binds to an E in the +1 position. This is different from several other reported LIR-LDS structures where R70 in LC3B binds to an acidic +7 residue in the LIR motif. However, R70 is conserved in all human ATG8s (R67 in GABARAP) and in many reported ATG8-LIR structures found to interact with an E in the +1 position, exactly as shown for the LC3B-BLM structure shown in Figure 3. The presented structure is therefore a very typical example rather than an exception to previously reported LIR motifs. The authors also select peptides with a so-called LIR+ motif, but this is a previously reported variant, and the alphafold model for the CTSL2 peptide in Figure 4 suggests a quite normal LIR-LDS interaction, except that W is docked into pocket 2.

    While the structural analysis of the BLM peptide bound to LC3B supports the importance of electrostatic interactions, it is not the primary basis for the statement regarding a “strong preference for negatively charged residues adjacent to the LIR.” Rather, this conclusion results from the sequence features enriched across the selected peptides in the HC-set, which revealed a consistent enrichment of negatively charged residues flanking the core motif and in the +4 position.

    With respect to the statement that the study “revealed exceptions to the reported LIR motif,” the intent was not to suggest that these interactions are unprecedented in the literature, but rather that we expanded the number of known exceptions to the most canonically defined LIR motif ([FWY]xx[LVI]). We do not claim these are unprecedented; we cite prior examples of exceptions in the text (Li 2018, Knævelsrud 2013, Chatzichristofi 2023). To avoid confusion, we revised the abstract to state that the study “revealed exceptions to the canonical LIR motif ([FWY]xx[LVI]).”

    (3) A limitation related to the search for binding determinants can be that the screen is only performed with LC3B. The impact of several determinants beyond the core LIR is known to be ATG8 specific. For instance, LC3-specific R10 and R11 are known to form electrostatic interactions with acidic residues in positions -2, -3, and -4 relative to core LIR, and the -3 and -4 positions can be a determinant for LC3 binding (Rogov et al, 2023; Autophagy Rep. 2: 2188523). Here, the authors made a consensus peptide based on the sequence logo derived from HC-set (Figure 3C). It is interesting that this peptide had a very high affinity for LC3B (0,1 µM) that was found to depend on the D in position -3. In the sequence logo shown in Figure 3A, D, and E are strongly favored in position -3, but position -4 is not included in the logo. Are acidic residues similarly enriched in the -4 position?

    The high affinity of the consensus peptide for LC3B is not dependent on the D in position -3. The peptides pCONSW-Nterm-S (EEDDDDEDWEILDIDEDEDGSGNEFYE) and pCONSW-Nterm-S-D-A(EEDDDAEDWEILDIDEDEDGSGNEFYE), where D in position -3 is mutated to A, have the same affinity (0.12 µM). The truncated peptide, pCONSW-Nterm-L (EDWEILDIDEDEDGSGNEFYE), which lacks position -3, has a weaker affinity of 0.66 ± 0.1 µM. These results illustrate that the flanking acidic residues, rather than specifically D in position -3, contribute to affinity (Figure 3C, Supplementary Table 3). The logo corresponding to the entire consensus peptide is shown in Supplementary Figure 5A. Acidic residues are not as enriched in the -4 position as they are in positions -1 through -3, although D is still the predominant residue in position -4 and is thus present in the consensus peptide.

    Extending this type of screen and analysis to other ATG8 family members has the potential to illuminate interesting paralog-specific differences.

    (4) Will the full list of peptides in the HC-set be included in a final manuscript? This is a very interesting set of peptides for scientists interested in LC3B-interacting proteins, and this includes those interested in noncanonical LIR interactions.

    Yes, all Supplementary Files (1-5) are available at Zenodo (10.5281/zenodo.17943925). Supplementary File 4 contains all of the peptides that were enriched through the sixth sort, and those peptides where “Average Z-score Across Sorts 4-6” is greater than or equal to (≥) 1.70 correspond to the full list of peptides in the HC-set.

    Minor comments:

    (5) When reading the result section, it is confusing that the bacterial membrane protein is named "circularly permuted OmpX" in the text on page 2, and "eCPX" in the figure legend. I suggest including both names in at least one place, as done in the methods section.

    We changed the text on page 2 to use the full name, which is “enhanced circularly permuted OmpX (eCPX)”, to address the confusion.

    (6) Figure S3A: The list of 10 peptides in the input library that have a KD in the LIRcentral database includes 7 with a KD below 5 µm (if I am correct), but is said to be 6 in the text on page 3.

    We corrected the text on page 3 to 7 peptides.

    (7) Error in the text. Data for the BLM peptide is shown in Figure 2B, but replaced by MAP1B in the text on page 3.

    We tested four peptides reported to co-immunoprecipitate with LC3B without an experimentally validated LIR motif (MAP1B823-858, ATG4A363-398, SCYL1640-675, and HEAT3377-412). Of these four, binding of three (ATG4A363-398, SCYL1640-675, and HEAT3377-412) is illustrated in Figure 2B, in addition to BLM552-587, which has not been previously reported to co-IP with LC3B. We added a note that not all curves are shown. Dissociation constants for these 5 peptides are included in Supplementary Table 1.

    Reviewer #2 (Recommendations for the authors):

    Human MAP1LC3B (LC3B) binds proteins involved in autophagy and other cellular processes using a short linear motif known as the LC3-interacting region (LIR).

    To discover peptides that interact with LC3B and profile the key binding determinants, the authors screened a library of ~500,000 36-residue peptides derived from the human proteome using bacterial cell-surface display. Analysis of the screening data, coupled with structural studies and site-directed mutagenesis, revealed exceptions to the reported LIR motif and a strong preference for negatively charged residues adjacent to the LIR.

    The authors also suggest that mutations in LC3B, shown to abrogate binding of LIR-containing peptides, instead alter LC3B binding specificity, leading to enhanced binding of some LIR-containing sequences.

    This is an interesting study, well structured, with solid data.

    My major concern is that the conclusion about the effect of known LC3B mutations on the binding of LIR-containing proteins is not strongly supported by the presented data. Did the authors try molecular dynamics simulations in such LC3B mutations to examine if F52A/L53A substitutions create alternative hydrophobic interfaces that modulate LIR motif peptide binding in a sequence-dependent manner?

    Figure 5C includes data that demonstrate that mutations to the LIR docking site (F52A, L53A) change the binding affinity of several peptides. Our structural analysis of the F52A/L53A LC3B mutants using ChimeraX (Figure 5A) provides a model for the changes in the hydrophobic pockets and a possible explanation for how these mutations introduce altered hydrophobic contacts and sequence specificity at the LDS. It would be challenging to exhaustively sample binding modes for peptides with different sequences using MD, which is best suited for exploring local minima. We do not claim to have a detailed structural explanation for the observed changes in binding specificity.

    Additionally, in vitro cellular data on the expression and localization of some of their strongest LC3B binding proteins (and their relevant novel residue mutations) would be useful.

    Assessing the expression and localization of our strongest LC3B-binding proteins and testing the effects of novel residue mutations in cells would be a valuable follow-up to further explore the implications of our findings. However, these experiments are beyond the scope of the current study, which focuses on high-throughput characterization of LC3B binding determinants using biochemical methods. We close the paper with a statement highlighting this as a promising direction for future investigation.

    Finally, the suggested refinement of the LIR motif sequence is not easily identifiable in the main text.

    We added a clause to clarify this in the Discussion: “Although a small number of LC3B-binding peptides bearing this LIR+ motif have been reported (~1% of cataloged hAtg8-binding peptides) (Li et al., 2018; Chatzichristofi et al., 2023), their prevalence appears underappreciated given their relatively high frequency in the HC-set (Supplementary Figure 10), leading to the refined motif [FWY]xx[LVIFWY].”

    Reviewer #3 (Recommendations for the authors):

    Please find my specific comments below.

    (1) The authors identify a large number of peptides from their screen, including several strong binders as shown in Figure 2C, that appear to bind LC3B but are from proteins that have not been previously annotated as LC3B binders in cells. Therefore, it would be helpful to validate whether some of these proteins are indeed LC3B binders in cells using CO-IPs or another related technique. This would significantly strengthen the manuscript, further validate their approach for characterizing LIR LC3B binding interactions, and demonstrate novel potential autophagy regulators.

    Validating candidate LC3B binders in cells using co-immunoprecipitation or related techniques will be a valuable follow-up and could provide important insight into novel autophagy regulators. Such experiments are beyond the scope of the current manuscript, which focuses on the high-throughput characterization of LC3B binding determinants in vitro. We mention this in the discussion as a promising avenue for future investigation.

    (2) In Figure 2C, there are two proteins that have high mean-Z scores but no detectable binding to LC3B (top right quadrant). It would be helpful for the authors to acknowledge this and speculate on why binders are isolated but cannot have detectable binding. Is this expected for a screen of this size? Is this below the non-specific binding expected or above?

    Some apparent false-positive binders may have been isolated and exhibit high mean Z-scores due to avidity effects from the pre-tetramerized LC3B used in the bacterial display screen. Binding was validated using monomeric LC3B in BLI experiments, which eliminates these multivalent effects. Consequently, weak interactions stabilized by LC3B tetramerization that enrich in the bacterial display assay may not be detectable at the monomeric LC3B concentrations tested in BLI. Given the differences in multimerization between the screening and validation assays, as well as the large size of the library, both false positives and false negatives are expected. We added a comment about the apparent false positives to section Strongly enriched peptides include novel candidate LC3B interactors.

    (3) In Figure 2C, it says four peptides are colored as in 1C, but I can only see three colored that way, and the yellow one is very faint and hard to see.

    We corrected the text in Figure 2C to three peptides and have darkened the yellow point for easier visualization.

    (4) The authors determine the crystal structure of a peptide from BLM bound to LC3B. However, no electron density is shown in the manuscript. Therefore, it is unclear how ordered the side chains are that are shown, including for D564-D566. The authors should include an electron density omit map for the BLM peptide so that readers can accurately interpret how ordered each side chain is in the peptide.

    Supplementary Figure 9 shows an ensemble of conformations consistent with the diffraction data, as well as an electron density omit map. Data to support visualization of the electron density for the structure is included with the PDB deposition (structure 9p3e).

    (5) In Figure 3C, the authors include numbers in the bar graph. What are the numbers that are included? Is this the mean Kd value? If so, the numbers may be misleading without a second significant figure since some values of 0.1 are far away from 0.2, while others are very close to 0.2. In addition, if these values are the Kd values, it would be helpful to include these values in all other bar graphs throughout the manuscript, including Figures 4A and 5C.

    The values reported are the mean KD values, in micromolar, which is now indicated in the legend of Figure 3C; we also added a second significant figure. The mean KD values have also been included for the bar graphs in Figures 4A and 5C.

    (6) On page 7, the authors state that "we found strong evidence for enrichment of LIR sequences, but not for these closely related motifs, indicating that whereas such LIR-related motifs may support binding in some sequence contexts, they are not sufficiently strong binding determinants to be enriched in our screen". I found this statement to be unclear. Do the authors mean that while LIR sequences were strongly enriched in the HC-set, LIR+ sequences were not? This statement should be clarified.

    The statement on page 7 is in reference to Supplementary Figure 10, which shows that peptides containing the canonical LIR [FWY]xx[LVI] are enriched in the HC-set relative to their presence in the input library. In contrast, peptides containing LIR-related motifs ([WFY]xx[WFY], [LVI]xx[FWY], and [LVI]xx[LVI]) are not strongly enriched in the HC-set relative to their presence in the input library. We clarified this statement to read “We found strong evidence that canonical LIR motifs ([FWY]xx[LVI]) are enriched in the HC-set relative to their frequency in the input library, whereas LIR-related motifs ([WFY]xx[WFY], [LVI]xx[FWY], [LVI]xx[LVI]) are not (Supplementary Figure 10), indicating that although LIR-related motifs can support LC3B binding in certain sequence contexts, they are not sufficiently strong determinants to drive enrichment in our screen.”

    (7) How different is the alphafold prediction of the LIR+ engaging with LC3 compared to the LIR LC3 interaction? Are they largely similar or are there unique features to each? Does the presence of a second large aromatic residue push the backbone of the peptide farther away from the hydrophobic binding pocket?

    To address this, we predicted structures of the LIR+ motif of CTSL2 (EEDWEVFDPY) and a canonical LIR version (EEDWEVIDPY) to LC3B using AlphaFold3. Structural superposition shows that LC3B adopts a conserved conformation in both complexes (Cα RMSD 1.55 Å). The backbones of the core LIR / LIR+ motif also align closely to each other (1.39 Å RMSD), indicating preservation of the canonical binding mode. The LIR+ peptide exhibits a modest increase (~2 Å) in Cα distance between the aromatic residue Phe and LC3B F52 in HP2 compared to the corresponding Ile in the canonical LIR (8.3 Å vs 6.3 Å). Thus, a subtle backbone repositioning is predicted to accommodate the aromatic side chain. We added a comment about this to the section Highly enriched peptides lacking a canonical LIR can bind LC3B.

  8. Version published to 10.64898/2026.01.05.697675v2 on bioRxiv
  9. Version published to 10.7554/elife.110318.1 on eLife
  10. eLife

    eLife Assessment

    This study is a valuable contribution that comprehensively identifies and characterizes LC3B-binding peptides through a bacterial cell-surface display screen covering approximately 500,000 human peptides. The data presented are solid, although this approach has limitations (e.g., it cannot assess the effects of post-translational modifications, which are often relevant to LIR-mediated interactions). Validation of the newly identified binding peptides by demonstrating their interactions with full-length proteins in cells would further strengthen this manuscript.

  11. eLife

    Reviewer #1 (Public review):

    Summary:

    This study uses high-throughput bacterial cell-surface display to identify LC3B-interacting peptides in the human proteome. The screen is unbiased, and this type of assay has not previously been used for selecting LC3B-interacting peptides. The screen was done with a library of 500,000 peptides, and they ended up with 427 peptides that they scored as high-confidence LC3B binders. The experiments performed are solid, and data are analyzed using well-documented methods and statistics.

    The aim of the authors was to isolate LC3B-interacting peptides from the human proteome, and the screen succeeded in doing so. The selected set of peptides included several previously reported LIR motifs, but also many novel LC3B binding peptides that either contained or did not contain the canonical core LIR motif [WFY]xx[LVI].

    Another aim was to identify binding determinants important for the LC3B interaction, and they made an interesting sequence logo based on selected LIR-containing peptides. However, this study does not really extend our knowledge related to binding determinants essential for LIR motifs in LC3B binding. They basically verify known characteristics, including the importance of varied types of electrostatic interactions supporting the docking of the core LIR into the LDS of LC3B.

    Strengths:

    The approach used here (high-throughput bacterial-surface-display) is new. The screen is unbiased, and the fact that peptides are directly tested for LC3B binding may facilitate the discovery of non-canonical LIR motifs. The screen appears to be highly selective and manages to distinguish between peptides that interact with LC3B and peptides that do not interact.

    Weaknesses:

    It is a limitation that no proteins are analyzed in this study. Further work is therefore needed to verify that identified LIR motifs are functional in full-length proteins and in cells.

  12. eLife

    Reviewer #2 (Public review):

    Summary:

    To discover peptides that interact with autophagy-related protein LC3B and profile the key binding determinants, the authors screened a library of ~500,000 36-residue peptides derived from the human proteome using bacterial cell-surface display. Analysis of the screening data revealed exceptions to the reported LIR motif and a strong preference for negatively charged residues adjacent to the LIR.
    These results support a refinement of the LIR motif definition and expand the network of candidate LC3B interaction partners.

    Strengths:

    High-throughput approach.

    Weaknesses:

    Lack of in vitro data and molecular dynamics simulations.

  13. eLife

    Reviewer #3 (Public review):

    Summary:

    The LC3 family of proteins, which includes LC3B, are ubiquitin-like proteins that are covalently linked to phosphatidylethanolamine in the expanding autophagosomal membrane during autophagy. LC3 family members bind to short sequences of amino acids that reside within dynamic regions in a wide variety of proteins. These sequences, termed LC3 Interacting Regions (LIRs), were initially thought to function primarily to link LIR-containing autophagy cargo receptors to LC3 family members to help facilitate their capture during autophagy. However, the functional importance of LIRs in autophagy has broadened to include more general functions in autophagy as well. While a general consensus for LIR sequences has been described as [FWY]0-X1-X2-[LVI]3, recent work has suggested that additional sequences outside of the canonical LIR sequence can bind LC3 family members and play important roles in autophagy. In this manuscript by Kosmatka et al, the authors perform a high-throughput screen using bacterial surface display coupled with fluorescence-associated cell sorting to identify which human sequences can bind to LC3B. They identify a variety of peptides capable of binding LC3B, including peptides from proteins that have not previously been described as LC3B-binding proteins. The results from the bacterial surface display were then used to guide sequence analysis, mutational analysis, and structural studies to further characterize the range of LIR sequences that are capable of binding LC3B. Taken together, this work adds to the growing knowledge of how LIR sequences interact with LC3 family members and demonstrates which amino acids both inside and outside of the LIR sequence aid in binding. This work also identifies new potential LC3 binding proteins, which may play unknown roles in autophagy regulation. Lastly, this work reinforces the importance of alternative LIR sequences such as the [WFY]0-X1-X2-[WFY]3 sequence, which the authors have dubbed the LIR+ sequence.

    Strengths:

    The manuscript uses a robust approach to identify and characterize different peptide sequences that can interact with LC3B. They validate a large number of sequences using biolayer interferometry (BLI) and attempt to correlate different amino acids with their binding affinity for LC3B. The large number of LC3B binding sequences and their dissociation constants adds significant new information to the field that will help others understand what sequences can bind to LC3B. The authors are also very careful to accurately report on their data and not overly interpret their findings.

    Weaknesses:

    After the authors identify proteins from their bacterial display assay, the remainder of the manuscript is focused on characterizing the different types of sequences that are identified in addition to validating the LC3B-LIR interactions using biochemical approaches, including BLI and X-ray crystallography. However, it's not entirely clear if the screen identified novel LC3B binders that interact with LC3B in cells. While I acknowledge that the focus of the manuscript is on the characterization of LIR sequences that can bind LC3B, it seems like a missed opportunity not to validate a few of the novel LC3B binders in vivo. This may result in the demonstration of novel binders of LC3B in cells and may further demonstrate the strength of this approach for identifying LC3 family member binding partners. Therefore, it would be helpful to look at a few proteins identified in the HC set that have not previously been identified as LC3B binders in cells to determine if they CO-IP with LC3B or interact with LC3B using a different approach.

  14. Mentioned by @biorxiv-biochem.bsky.social on Bluesky

    A proteome-wide biochemical screen defines binding determinants of the core autophagy protein LC3B https://www.biorxiv.org/content/10.64898/2026.01.05.697675v1

  15. Mentioned by @biorxivpreprint.bsky.social on Bluesky

    A proteome-wide biochemical screen defines binding determinants of the core autophagy protein LC3B https://www.biorxiv.org/content/10.64898/2026.01.05.697675v1

  16. Version published to 10.64898/2026.01.05.697675v1 on bioRxiv