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  1. Evaluation Summary:

    This manuscript describes follow-up studies on a hit from a proteome-wide screen for peptides that can bind to the EVH1 domain of the ENAH protein, one of three highly similar Ena/VASP actin regulators. The hit investigated is from a protein called PCARE, which selectively binds to ENAH but not the other two members of the Ena/VASP family, EVL and VASP. The authors provide a good explanation for how this selectivity is achieved and develop a peptide, PCARE-Dual, that specifically binds ENAH more tightly, setting out the stage for developing potent and selective inhibitors of ENAH activity.

    (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 name with the authors.)

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  2. Reviewer #1 (Public Review):

    The Ena/VASP family of actin regulators contain three highly homologous proteins, ENAH, EVL, and VASP. They have partially overlapping functions but also has unique functions. They use and EVH1 domain to bind many partner proteins to regulate the actin cytoskeleton. How binding specificity is determined is not well known. Here, the authors characterized one peptide binder, derived from a protein called PCARE. This peptide binder is a hit from proteome-wide screen that the authors report in a separate manuscript. Surprisingly, the PCARE peptided binds ENAH with 0.19 uM Kd and is very selective for ENAH over VASP and EVL. Using X-ray crystal structure and mutagenesis, the authors provide a very nice explanation for how the binding specificity is achieved, which is not trivial. Furthermore, using the understanding of the binding mode and specificity, the authors developed a peptide, PCARE-Dual, with a Kd of 50 nM for ENAH, which could be a useful research tool for studying the functions of ENAH.

    Overall, this is very interesting work with many strengths. The selectivity of PCARE for ENAH over VASP and EVL is very interesting. The FP4 motif binds EVH1 in the opposite orientation is interesting. The 14-residue C-terminal to the FP4 motif make additional contacts to EVH1, explaining its high affinity. The binding-induced conformation change in ENAH nicely explained the specificity of PCARE for ENAH over VASP and EVL. Using modeling method dTERMen to come out with a mutant of EVL that binds to PCARE B with 0.35 uM Kd is very interesting. Getting a peptide PCARE-Dual with 50 nM affinity to ENAH and selective for ENAH is impressive.

    The reviewer only found a few weaknesses/questions, which are relatively minor and can be addressed by better explanation or minimum experiments:

    Even though PCARE peptide is strong binder for ENAH, whether the endogenous PCARE protein binds to ENAH or not was not demonstrated. Even though this is not directly relevant for the main conclusion of the manuscript, the discussion of the manuscript does hint on the physiological function of this interaction. Thus, the protein-protein interaction should be validated in cells.

    "Mito-PCARE B serves to sink ENAH away from its normal sites of action" may not be correct, as there are still a lot of mito-PCARE B and ENAH near the plasma membrane based on Figure 2F.

    Why fusing the 36-mer PCARE to EVH1? What happened with the peptide not fused to EVH1?

    The authors used EVL V65P Y62C and showed that this mutant binds PCARE B 10-fold better than WT. How about the EVL Y62C mutant? Do you really need the V65P mutation?

    The authors envision that PCARE and PCARE-Dual could be promising leads for developing therapies to treat ENAH-dependent diseases, but this could be difficult to achieve in practice as it is likely going to be very difficult to shorten such long peptides while maintaining the potency and specificity.

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  3. Reviewer #2 (Public Review):

    Hwang et al. reports an interesting study that elucidates the structural mechanism by which only one of the three Ena/VASP paralogs, ENAH, selectively and tightly binds via its EVH1 domain to a newly discovered cellular target, PCARE. Peptides with enhanced affinities were generated by adding a second motif to contact a previously identified secondary binding site on the opposite face of the EVH1 domain. The conclusions of the paper are supported by the data.

    Strengths:

    Important findings include the identification of PCARE (photoreceptor cilium actin regulator) as a high affinity ENAH-specific cellular binding partner. PCARE is a vision-related protein involved in photoreceptor cell maintenance. How PCARE achieves its high affinity is revealed in the high-resolution crystal structure of ENAH EVH1 fused at its C-terminus to the PCARE peptide. This structure shows that the peptide binds in reversed N-to-C orientation and displays contacts beyond the canonical binding surface. How PCARE selectively binds tightly to EVAH but not to the close paralogs VASP and EVL was a surprise, also revealed by this structure. A loop fully conserved in all three proteins adopts a different conformation in EVAH due to a surrounding network of non-conserved residues. Application of the computational tool dTERMen was able to identify this network and guided a series of mutations in the EVL EVH1 to definitively demonstrate these specificity determinants imparted high affinity binding to the EVL paralog.

    The PCARE peptide affinity was further enhanced by fusing to its C-terminus a linker and a second motif designed to engage with a previously characterized secondary binding site on the opposite side of the EVH1 domain of VASP EVH1. This secondary site is conserved in EVAH, and indeed the dual-motif PCARE-DUAL peptide bound with nanomolar affinity to EVAH EVH1 domain.

    Weaknesses:

    The structural conclusions are based on the crystal structure of a fusion protein, where the C-terminus of EVAH EVH1 is fused to the PCARE peptide. The main concern is that tethering the peptide to the C-terminus could induce the peptide to bind in the opposite orientation from the canonical binding interaction.

    The function of PCARE in the cell, and the relevance of its specific and tight interaction with EVAH is not explored. Interestingly, there is other recent literature that connects PCARE to actin dynamics.

    The design of PCARE-DUAL and other dual-motif ligands utilized in this study was based on work by another group that characterized the secondary binding site (Acevedo et al., 2017, "A Noncanonical Binding Site in the EVH1 Domain of Vasodilator-Stimulated Phosphoprotein Regulates Its Interactions with the Proline Rich Region of Zyxin", Biochemistry 56(35):4626-4636, doi: 10.1021/acs.biochem.7b00618). This prior work was not credited as the foundation on which their affinity enhancement strategy was built.

    An additional key finding of the Acevedo 2017 paper was that ligand binding to the novel secondary site is inhibited by the phosphorylation-mimicking mutation of Y39 to E, a site known to be phosphorylated by Abl. Since ENAH has Y at the equivalent site and is a reported target of Abl, the bivalent advantage the authors have designed into their dual-motif ligands might be disrupted by phosphorylation. This has implications for their proposed use of dual-motif ligands for "dissecting specific Ena/VASP functions in processes including cancer cell invasion" and "for developing therapies to treat ENAH-dependent diseases."

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  4. Reviewer #3 (Public Review):

    In the accompanying manuscript, the MassTitr method for profiling short linear motifs on a proteome-wide scale is described and identifies PCARE as a high affinity binding partner for EVAH EVH1 domain. The unique observation is that contextural sequence surrounding the polyproline motif in PCARE underlies the specificity for its interaction affinity relative to other binding partners, in that a reverse-peptide binding orientation is favored. While this result may, in isolation, be perhaps significant only to a subset of protein chemists, when combined with the description of MassTitr development, it provides an additional example of how the methodology can uncover non-canonical binding interactions.

    Strengths:

    The work provides another powerful example of how the MassTitr method can identify non-obvious binding partners with unique interaction features. The biochemical studies that were employed to elucidate the mechanism for this specificity strongly support the conclusions.

    Weaknesses:

    These results in isolation may impact only a limited number of protein chemists working on this or related systems; however, when coupled with the general description of the MassTitr method, the impact is much broader.

    One of the EVL mutants that was generated and characterized was Y62C. This variant has the capacity to form inter-chain disulfide bonds since the newly incorporated Cys is unpaired, but this possibility is not discussed or examined.

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