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

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.