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  1. Author Response

    Reviewer #3 (Public Review):

    This paper by Goodman et al. is the latest in a series focusing on the structural determinants of clustered protocadherin (cPcdh) isoform cis- and trans-interactions. The goal of this particular paper is to garner further details in support of the "isoform-mismatch chain-termination model" of cPcdh interaction, which was developed by the group in 2015. The model is based on their landmark initial crystallographic structural analysis of particular cPcdh ectodomains, as well as on earlier work from other groups showing that (at least) some cPcdh proteins interact homophilically in trans but promiscuously in cis. The model predicts that cis-dimers of various cPcdh isoforms form via the 5th and 6th extracellular cadherin repeats (EC5/6), and that these dimers then interact in trans strictly heterophilically via EC1-4 to form "dimers of dimers" as an initial event. If cPcdh repertoire between two cells primarily matches, then a linear "zipper" of such dimers will expand, increasing interaction and presumably associated intracellular signaling. Mismatching isoforms expressed in one cell but not the other will terminate this zipper chain, and thus cPcdh repertoire matching between cells will determine self/non-self recognition. Other groups have shown that homophilic matching between neurons is-depending upon the neuronal subtype-important for driving neurite self-avoidance or growth and branching of dendritic arbors, so the mechanisms of interaction will be important to understanding events in neural development.

    The present paper builds on others by the group (e.g., Rubinstein et al., 2015, Goodman et al., 2016, 2017, Brasch et al., 2019), and primarily extends these results to more isoforms, providing also more molecular detail. There are three main findings. First, the concept that cPcdh trans-interactions are strictly homophilic is supported by many new analyses using surface plasmon resonance (SPR) assays in which an ectodomain of one isoform is coupled to a chip and those of identical vs. distinct isoforms are flowed over it to measure interactions. The data are rigorous and nicely presented and demonstrate-unsurprisingly given many prior demonstrations-that trans interactions mediated by EC1-4 are strictly homophilic. A main advance here is in the methodology, which can quantitatively and directly measure such interactions, in contrast to the qualitative cell aggregation studies that were already published. The authors also present an informative mutagenesis series identifying 5 interfacial residues that, when mutated individually or in concert to match a different highly similar intra-family isoform quantitatively shift trans-interaction from homo- to heterophilic.

    The second main finding is the presentation of a new antiparallel trans-dimer structure of the gC4 EC1-4 interaction. While structures of other gamma Pcdhs have been published by the group before, the addition of the C4 structure is important for several reasons: 1) this isoform is the only one of all the cPcdhs that is essential for postnatal viability and normal neuronal survival in mice; 2) this isoform is the only one of the gamma Pcdh family that does not make it to the plasma membrane without dimerizing with a "carrier" cPcdh of some kind, which had cast doubt on whether it would interact in the same way as other cPcdhs; 3) A recent publication (not cited by the authors yet as it came out coincident with their submission) demonstrated that truncating or structure-disrupting mutations in the human PCDHGC4 gene result in significant neurodevelopmental disorders. The authors show that the structure of the C4 trans-dimer is similar to that found for other cPcdh isoforms, though the interaction is weaker than observed for others. They suggest that particular residues in the EC1:EC4 and EC2:EC3 trans interface may be responsible for this, though they do not follow up with mutation experiments to confirm. Doing so (mutating the identified C4 residues to those of, say gB2 or a delta2 Pcdh) would contribute to the novelty of the paper, as it is unclear as of yet how strength of cPcdh interactions might be regulated or manipulated.

    We thank the reviewer for drawing our attention to the missing citation for the recent paper on PCDHGC4 variants implicated in neurodevelopmental disorders (Iqbal et al. 2021), which we have now added. We have performed the requested experiments and, as now discussed in the manuscript, they validate the role of E78 but not D290 in significantly weakening the dimerization of γC4.

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  2. Evaluation Summary:

    This paper is of interest to cell biologists, biochemists and biophysicists interested in how adhesion and signaling proteins at the cell surface help cells (and especially neurons) interact and perform self/non-self-recognition and self-avoidance. The authors provide the first extensive biophysical dataset examining a large subset of potential trans (across two cells) and cis (on the surface of the same cell) interactions between different isoforms of the ~60 clustered protocadherins (cPcdhs). There data show that all tested trans interactions are strictly homophilic and that not all possible cis interactions are equivalent. These results provide additional layers of complexity and constraints on how this protein family can provide neurons with the ability to perform self-recognition and self-avoidance.

    (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 #2 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    The manuscript is of interest to those studying the biophysical rules of adhesion molecules, and those studying the molecular underpinnings of synaptic self-avoidance. The work adds much of the biophysical detail that existing model lacked, and therefore provides support to the prevailing mechanism. The data is of high quality and interpreted properly.

    Protocadherins are the proposed receptors for self-avoidance in vertebrate neurons. The authors have put forth an "isoform-mismatch chain-termination model" in previous manuscripts with ample structural evidence to support it, and further advance this model in current paper by providing a large amount of data on interaction biophysics. The paper strengthens previous claims that trans interactions (between cells) are only homodimeric, but also show that cis interactions (on the same cell) are promiscuous. They present specific reasons to why and how a few cis pairs form in asymmetric patterns. The cis interactions (if they exist and the exact asymmetrical nature) do not appear to follow a strong general rule (there is a preference for pairs from non-matching classes) - but they do not have to.

    In the clustered protocadherin system, one of the unknowns was relative affinities and promiscuities of the homophilic vs heterophilic interactions. The authors report SPR data for 100+ heterophilic (trans) cPcdh interactions, showing strict homodimerization but no interactions between non-identical pairs. The binary nature of these interactions (present or absent), as the authors highlight, is indeed very remarkable. The crucial biophysical measurement of these affinities are provided by AUC data, although the data are not provided - only calculated dissociation constants in a supplemental table are. They report crystal structures of a C-type protocadherin (gC4 domains 1 to 4), which generally agrees with previous reports of cPcdh structures. They also test which cPcdh forms which side of the cis dimer via MALS by mutating the interfaces looking for heterodimer formation.

    Overall, the manuscript sets out to answer a specific, limited set of questions, and does that very well. The amount of biochemical work is immense, and the interpretation of the data is masterly. (There are technical limitations to measuring affinities in mixed homo and heterodimeric systems, which prevents the authors from drawing a complete energetic description of the system, but it is not clear if one is needed to understand the relevant biology, or even sufficient to model it.) The reported structures are of superior quality, and even the lower resolution, anisotropic dataset reported is, if anything, undersold. It is clear that very careful model building has been performed in real space. There are only minor issues that the authors may want to address.

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  4. Reviewer #2 (Public Review):

    Overall, the data are solid, extensive, well-illustrated, and well-presented. This is the first extensive biophysical analysis, and provides very useful quantitative data in considering how cPcdhs work. The conclusions are overall well supported by the data. The paper can be divided into three important sets of results:

    First, the authors use SPR to demonstrate that all tested trans dimerization interactions are strictly homophilic. The authors emphasize that this is the strictest homophilic preference observed thus far for a family of adhesion proteins. It remains unclear whether or how this strict homophilic specificity could be important for the proper function of the cPcdh protein family.

    Second, the authors present two crystal structures of a C-type trans interface (that of γC41-4), the first structures of available for a C-type cPcdh. They find that it is similar to the trans interfaces observed for both clustered and non-clustered protocadherins. Therefore, even though C-type cPcdhs have distinct expression patterns and significant sequence divergence compared to other cPcdhs, their interaction architecture is effectively the same. This is important in considering how the C-type cPcdhs could be incorporated just like other isoforms in the large zipper assemblies that are postulated at cell-cell encounters by the chain termination model for self-recognition.

    Third, the authors measure the binding affinity a significant number of both homophilic and heterophilic cis dimerization interactions and find them to be not indiscriminately promiscuous, but rather in many instances preferentially heterophilic over homophilic. This is surprising, and the available data can start to provide additional constraints on the chain termination model.

    The first two advances provide support for a simple model for cPcdh assemblies: we can safely only consider trans homodimers (no trans heteromers), and we can consider all trans interfaces to be roughly equivalent in terms of protein architecture (within a few angstroms of RMSD). The third advance, in contrast, suggest that we should incorporate some additional constraints on possible cis interfaces, beyond the previously observed constraints that alpha isoforms can only form cis heterodimers. Although the authors provide several interesting discussion points about how to consider the new cis interface information, they do not go as far as to develop an updated chain termination model that incorporates the information - this will likely take some time and effort, and perhaps additional data and information.

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  5. Reviewer #3 (Public Review):

    This paper by Goodman et al. is the latest in a series focusing on the structural determinants of clustered protocadherin (cPcdh) isoform cis- and trans-interactions. The goal of this particular paper is to garner further details in support of the "isoform-mismatch chain-termination model" of cPcdh interaction, which was developed by the group in 2015. The model is based on their landmark initial crystallographic structural analysis of particular cPcdh ectodomains, as well as on earlier work from other groups showing that (at least) some cPcdh proteins interact homophilically in trans but promiscuously in cis. The model predicts that cis-dimers of various cPcdh isoforms form via the 5th and 6th extracellular cadherin repeats (EC5/6), and that these dimers then interact in trans strictly heterophilically via EC1-4 to form "dimers of dimers" as an initial event. If cPcdh repertoire between two cells primarily matches, then a linear "zipper" of such dimers will expand, increasing interaction and presumably associated intracellular signaling. Mismatching isoforms expressed in one cell but not the other will terminate this zipper chain, and thus cPcdh repertoire matching between cells will determine self/non-self recognition. Other groups have shown that homophilic matching between neurons is-depending upon the neuronal subtype-important for driving neurite self-avoidance or growth and branching of dendritic arbors, so the mechanisms of interaction will be important to understanding events in neural development.

    The present paper builds on others by the group (e.g., Rubinstein et al., 2015, Goodman et al., 2016, 2017, Brasch et al., 2019), and primarily extends these results to more isoforms, providing also more molecular detail. There are three main findings. First, the concept that cPcdh trans-interactions are strictly homophilic is supported by many new analyses using surface plasmon resonance (SPR) assays in which an ectodomain of one isoform is coupled to a chip and those of identical vs. distinct isoforms are flowed over it to measure interactions. The data are rigorous and nicely presented and demonstrate-unsurprisingly given many prior demonstrations-that trans interactions mediated by EC1-4 are strictly homophilic. A main advance here is in the methodology, which can quantitatively and directly measure such interactions, in contrast to the qualitative cell aggregation studies that were already published. The authors also present an informative mutagenesis series identifying 5 interfacial residues that, when mutated individually or in concert to match a different highly similar intra-family isoform quantitatively shift trans-interaction from homo- to heterophilic.

    The second main finding is the presentation of a new antiparallel trans-dimer structure of the gC4 EC1-4 interaction. While structures of other gamma Pcdhs have been published by the group before, the addition of the C4 structure is important for several reasons: 1) this isoform is the only one of all the cPcdhs that is essential for postnatal viability and normal neuronal survival in mice; 2) this isoform is the only one of the gamma Pcdh family that does not make it to the plasma membrane without dimerizing with a "carrier" cPcdh of some kind, which had cast doubt on whether it would interact in the same way as other cPcdhs; 3) A recent publication (not cited by the authors yet as it came out coincident with their submission) demonstrated that truncating or structure-disrupting mutations in the human PCDHGC4 gene result in significant neurodevelopmental disorders. The authors show that the structure of the C4 trans-dimer is similar to that found for other cPcdh isoforms, though the interaction is weaker than observed for others. They suggest that particular residues in the EC1:EC4 and EC2:EC3 trans interface may be responsible for this, though they do not follow up with mutation experiments to confirm. Doing so (mutating the identified C4 residues to those of, say gB2 or a delta2 Pcdh) would contribute to the novelty of the paper, as it is unclear as of yet how strength of cPcdh interactions might be regulated or manipulated.

    Finally, the authors extend and confirm that cPcdh cis-interactions are promiscuous between isoforms without being ubiquitous. The essential aspect of this finding has been known since 2010 and was confirmed in papers from this group in 2015, 2016, and 2017. The primary advance again is the use of SPR rather than cell aggregation or analytical ultracentrifugation, this time using EC3-6 constructs that contain the cis-interaction EC5/6 interfaces. The data support that cis-interactions are, at least, more promiscuous than are the strictly homophilic trans-interactions, but do reveal more about the "rules" governing which isoform can interact with which others. The key finding is that interfamily (e.g., beta Pcdhs with gBs, or C-type with betas, gA or gB) heterodimers are favored and that homodimers are disfavored (with intra-family heterodimers occurring rarely). The authors complete the study by demonstrating nicely that gA4 preferentially plays the "EC6 only" part of a cis-dimer with gC3, which plays the "EC5/6" part; this builds on prior results from Goodman et al., 2017 showing this slightly offset method of cis-dimerization whereby the EC6 of one partner interfaces with EC5 and 6 from the other. Evidence is presented that at least some isoforms exhibit "handedness" in terms of which role they will play in a dimer, which is important in that it would limit the dimers that could efficiently form and thus have implications for the model they have been building. Some nice mutational studies are presented confirming this by SPR (mutations that block an isoform from an EC6 position or an EC5/6 position but not vice-versa).

    The data are generally well-presented and rigorously collected; though this reviewer is not a crystallographer the presentation of the structures is clear and all supportive data are present. They support the conclusions drawn and are logical. The main issue with the paper is that, as noted above, the advances are somewhat incremental in that the main points were known prior to this study: the cPcdhs interact in trans in a strictly homophilic manner (first shown in 2010 for some isoforms, then 2014 for all, and later in 2015 and 2016 in structures); individual isoforms interact in trans in an antiparallel manner involving EC1-4 (C4 structure is new but the main conclusion is unaltered from the other structures reported in 2015 and 2016); and cPcdh cis interactions are promiscuous but constrained by some partner preferences (first reported as promiscuous based on a few isoforms in 2010, 2014, and later confirmed but with exceptions and mechanistic detail in 2016 and 2017). What is new is the methodology, which in some cases is more direct and open to quantitative determinations; the type and number of examples collected; and the aforementioned mutational analyses that drive home the conclusions Still, the paper is largely confirmatory of the authors' many excellent prior papers. For example, in Goodman et al., 2017 (PNAS), the authors present structures yielding a putative EC5/6+EC6 dimer surface and state "the structure explains the known restrictions of cis-interactions of some Pcdh isoforms"-these restrictions were known due to Goodman et al., 2016 (eLife) where it was shown that gA's do not homo-dimerize in cis but can hetero-dimerize with alpha Pcdhs. Because of this, the impact of this latest work is likely to be greatest for researchers who directly work on the Pcdhs or other members of the cadherin superfamily, for whom the additional data will be welcome confirmation and extension. Due to the confirmatory nature of the paper the impact may be less apparent to the more general reader.

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