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  1. Author Response:

    Reviewer #1:

    The weaknesses of the manuscript include:

    (1) Among the 108 binders selected, the authors further limited the 108 binders to 33 after bioinformatics analysis among which 14 are previously known to interact or co-localize with an Ena/VASP protein. However, whether the new ones really bind to ENAH is not tested.

    After reanalysis of the literature, we realized that NHSL1 has not previously been validated to bind to Ena/VASP proteins, although it shares many common binding proteins. This has been updated in the text, Figure 1E, and Supplementary File 1. Thus, we validated seven hits previously not reported to bind to ENAH using biolayer interferometry assays for which the data are included in Supplementary File 2, and three by co-IP in mammalian cells.

    (2) The structure analysis for EVH1 of ENAH is nice, but inference to specificity was not tested by mutagenesis.

    Paralog specificity, although very interesting to us, is a peripheral point in this particular paper and we have not tested our hypotheses about ENAH vs. VASP or EVL specificity by mutational analysis. We now point this out in the text, where we report our observations and include a discussion of possible mechanisms but do not claim that we have established the origins of specificity between these paralogs. Our most interesting finding about paralog specificity of hits from this screen is elaborated in a separate manuscript that addresses the highly selective binder PCARE; this is now published.

    (3) Although the MassTitr method works nicely, it is not easily apparent how much new insights the study has provided from the MassTitr screening. Perhaps the additional proline is one new insight. The double FP4 motif seems to be already known in the literature.

    The past decade has seen a rise in screening approaches to identify consensus short linear motifs for important signaling proteins. However, our study is the first to comprehensively map local and distal sequence elements surrounding a SLiM. MassTitr was useful for this purpose because it enabled us to screen longer peptides and identify high-affinity binders from the screening output. This allowed us to interrogate how context effects modulate affinity to a given SLiM-binding domain. In this Short Report we highlight how our method makes the role of context readily apparent in the screening results. This is a general approach. In this work, it provided multiple hypotheses, several of which we followed up using biophysical studies.

    The double FP4 motif was previously uncovered in detailed NMR studies of the interaction of zyxin with VASP (Acevedo et al., 2017). Here we show that multiple human proteins that bind to ENAH EVH1 domain contain such dual motifs, that there appears to be a preferential spacing between the motifs (Figure 3A), and that this pattern confers a binding advantage for ENAH, where the existence of a back- side side could be postulated based on study of VASP but has not been previously tested. We further illuminate a preference for C-terminal proline residues, and for flanking negative charge, and we found a protein (PCARE) that uses flanking sequence to achieve unprecedented affinity for ENAH.

    Our screening also uncovered previously unknown binders of ENAH, several of which we validated biochemically and in cells. Even among previously characterized binders pulled from our screen, the exact Ena/VASP binding site was previously unknown. Our MassTitr screen identified novel ENAH EVH1 domain binding sites for 23 previously characterized and unknown binders of the Ena/VASP family. Many of these binders link Ena/VASP proteins to new and emerging biology, such as highlighting an underappreciated role for ENAH in cilia. Furthermore, although this is only a Short Report, we provide a thermodynamic and mutational analysis of dual-motif peptides, and we provide a crystal structure that illuminates the origin of the preference for flanking proline residues.

    Our paper is an example of how a proteome-wide screen of long peptides can provide hints, in the motif contexts of the hits, that enable biophysical dissection of binding determinants for domains involved in important regulatory processes.

    We have revised the abstract and provided additional comments throughout the text to highlight the logic and outcomes of this screen.

    Reviewer #2:

    Weaknesses:

    The claim that the reported studies show "that proteins with two EVH1-binding SLiMs can wrap around a single domain" is overstated. No structural data is presented to show that dual ligands "wrap around a single domain". It is a logical possibility that is consistent with the data presented, and should be described as such.

    We agree with this and we have adjusted the language in the abstract. Our other mentions of this model throughout the paper were more circumspect, emphasizing the consistency of our data with this model. The Discussion contains an expanded discussion of our observations and their possible interpretations.

    To explain why lengthening the linker between two binding motifs in a dual ligand weakens binding by only 2-fold, the authors suggest that the linker may interact favorably with the non canonical site. A likely alternative explanation is the effect of linker length on the effective concentration, Ceff, which reflects the concentration of a second binding motif after the first motif is bound.

    We expect that the effective concentration is an important factor contributing to the affinity of dual-motif peptides, as suggested by the reviewer. Our proposal that the linker may interact with the domain was based on our observation that the peptides that were truncated to include only one motif (i.e., NHSL1 FP4 1, NHSL1 FP4 2, LPP FP4 1, and LPP FP4 2) each showed at least 2-fold weaker binding to ENAH EVH1 R47A (which disrupts the back-side site) than to the WT EVH1 domain, suggesting that linker residues themselves and not just motif residues may be able to engage this site. But we agree that the linker length could also modulate affinity by changing the effective concentration for a multivalent interaction. We have elaborated our discussion of possible models to address this point and point #1 above. We suspect that there may be multiple modes in which peptides engage EVH1, influenced by the flanking sequence of the motif(s), and that these may change as truncations are made. A detailed NMR analysis would be required to provide a higher resolution model and is beyond the scope of this study.

    Based on the materials and methods, the authors appear to have employed an "ENAH tetramer construct" (human EVH1 fused to ENAH mouse coiled coil) for all of the reported experimental interaction studies. However, the ITC methods describe sample preparation with "ENAH EVH1 domain". The monomer versus tetramer has important implications for interpretation of the ITC data, since a tetramer introduces potential inter-domain binding by dual ligands. Clarification is needed to be able to evaluate fully the implications of the data.

    Thank you for bringing to our attention this omission in the details of our description. The ITC data were collected using a monomeric EVH1 domain construct, to avoid the problems highlighted by the reviewer. We have updated the manuscript to clarify when a tetrameric vs. monomeric ENAH EVH1 domain was used for each experiment.

    Reviewer #3:

    Weaknesses:

    In several cases, comparisons are made between KD values that are relatively similar (e.g., 3-fold or less). Additional analysis is required to demonstrate that these differences fall outside the range of experimental error. In addition, the Methods appear to be missing description of the BLI assay even though it is referred to in the text of the Results.

    We now present statistical tests for quantitative comparisons. We refer to the exact biolayer interferometry protocol that we used in our recently published eLife Research Article, Hwang et al. 2021 for full experimental details, but we have also now included a shorter description of our protocol in the methods section.

    The description of the selection and screening procedure in the Results is vague and lacks critical details.

    Details have been added to the methods section, particularly regarding the binning procedure (as requested). This is also supported by Figure 1 – figure supplement 4.

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  2. Evaluation Summary:

    The manuscript introduces a new molecular screen, MassTitr, to screen for long (36-mer) peptides derived from the human proteome that can bind a specific target. The method is demonstrated using the EVH1 domain of the actin-associated ENAH protein as target. About 100 peptides were isolated, and further analysis identified sequence features that contribute to the binding of the EVH1 domain by these peptides. The human proteome contains many short linear motifs of 4-6 residues that are critical for protein-protein interactions. The work here helps to better understand how the sequence surrounding such motifs contributes to protein-protein interactions.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

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  3. Reviewer #1 (Public Review):

    The manuscript uses a new screen called MassTitr, a method that is based on fluorescence-activated cell sorting (FACS) of a library of peptide-displaying bacteria and subsequent deconvolution of signals by deep sequencing. This method allows the authors to display long (36-mer) peptides derived from human proteome to screen for peptides that can bind the EVH1 domain of ENAH protein. About 100 peptides were identified and further analysis identified sequence features that contribute to the binding of EVH1 domain, including an additional proline after the FP4 motif and double FP4 motif.

    The strengths of the manuscript are: (1) the MassTitr method is very nice and has advantageous features, which allow the screening of longer peptides to explore sequences surrounding the FP4 motif (2) By screening the entire human proteome-derived peptides (416,611 36-mer peptides with 7-residue overlaps), many EVH1 binders have been identified, including previously unknown ones that do not contain an FP4 motif. (3) A nice structural explanation for why EVH1 of ENAH prefers FP4 motif with an additional proline is provided. (4) Mutational studies have nicely confirmed that the double FP4 motif contributes to stronger binding. (5) The thermodynamic analysis (entropy versus enthalpy contribution) of binding is interesting and makes sense.

    The weaknesses of the manuscript include: (1) Among the 108 binders selected, the authors further limited the 108 binders to 33 after bioinformatics analysis among which 14 are previously known to interact or co-localize with an Ena/VASP protein. However, whether the new ones really bind to ENAH is not tested. (2) The structure analysis for EVH1 of ENAH is nice, but inference to specificity was not tested by mutagenesis. (3) Although the MassTitr method works nicely, it is not easily apparent how much new insights the study has provided from the MassTitr screening. Perhaps the additional proline is one new insight. The double FP4 motif seems to be already known in the literature.

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  4. Reviewer #2 (Public Review):

    Hwang et al. reports an exciting study that screened a library of 36-residue peptides that span the entire human protein coding space to identify binding partners of a key interaction domain that regulates cytoskeletal remodeling. Novel binding partners were identified and regions of the ligands beyond the canonical binding motif were shown to play critical roles in determining specificity and affinity.

    The conclusions of the paper are mostly supported by the data, but clarifications of experimental details are needed as well as qualifications of some claims.

    Strengths:
    A powerful high-throughput screening of a peptide library was applied to search for binding partners of the EVAH EVH1 domain. Known binding partners provided controls, and new binding partners were identified. The longer length of peptides (36 amino acid residues) provided the opportunity to detect multivalent interactions. Indeed, many of the identified hits contained multiple EVH1 binding motifs.

    Complementary techniques were applied to assess the relevance of hits and to select a subset for experimental validation.

    Biophysical studies were employed to carefully quantify the most interesting interactions that were identified.

    Weaknesses:

    The claim that the reported studies show "that proteins with two EVH1-binding SLiMs can wrap around a single domain" is overstated. No structural data is presented to show that dual ligands "wrap around a single domain". It is a logical possibility that is consistent with the data presented, and should be described as such.

    To explain why lengthening the linker between two binding motifs in a dual ligand weakens binding by only 2-fold, the authors suggest that the linker may interact favorably with the non canonical site. A likely alternative explanation is the effect of linker length on the effective concentration, Ceff, which reflects the concentration of a second binding motif after the first motif is bound.

    Based on the materials and methods, the authors appear to have employed an "ENAH tetramer construct" (human EVH1 fused to ENAH mouse coiled coil) for all of the reported experimental interaction studies. However, the ITC methods describe sample preparation with "ENAH EVH1 domain". The monomer versus tetramer has important implications for interpretation of the ITC data, since a tetramer introduces potential inter-domain binding by dual ligands. Clarification is needed to be able to evaluate fully the implications of the data.

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  5. Reviewer #3 (Public Review):

    The manuscript by Hwang et al. establishes a new technique for proteome-wide screening of interacting SLiMs via bacterial display (MassTitr). Results from this screening analysis reveals non-obvious influence that context can play in specificity of interaction for polyproline-containing motifs. Specifically, MassTitr revealed an unusual FP8 motif that is not precedented for binding to the ENAH EVH1 domain, and the presence of repeating FP4 motifs from the screen provided evidence that a second non-canonical FP4 binding site may exist, again a novel finding that was enabled by the MassTitr analysis.

    Strengths: The MassTitr methodology appears to be straightforward and reliable, and thus should be of broad interest to protein and peptide chemists and potentially implementable in other laboratories. The identification of non-obvious context contributions identified for ENAH EVH1 domain provides an important example of how recognition likely includes more than the simple minimal motifs that are widely studied. Since such SLiM recognition proteins are widely distributed and important for a number of critical biological effects, this research is also of fundamental biological significance as well.

    Weaknesses: While the specific examples provided do highlight the significance of the MassTitr screening, these would be burnished with additional examples such as those described in the accompanying report.

    In several cases, comparisons are made between KD values that are relatively similar (e.g., 3-fold or less). Additional analysis is required to demonstrate that these differences fall outside the range of experimental error. In addition, the Methods appear to be missing description of the BLI assay even though it is referred to in the text of the Results.

    The description of the selection and screening procedure in the Results is vague and lacks critical details.

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