Uncovering the BIN1-SH3 interactome underpinning centronuclear myopathy

Reviewer #1 (Public Review):

Original review:
The authors report here interesting data on the interactions mediated by the SH3 domain of BIN1 that expand our knowledge on the role of the SH3 domain of BIN1 in terms of mediating specific interactions with a potentially high number of proteins and how variants in this region alter or prevent these protein-protein interactions. These data provide useful information that will certainly help to further dissect the networks of proteins that are altered in some human myopathies as well as the mechanisms that govern the correct physiological activity of muscle cells.

The work is mostly based on improved biochemical techniques to measure protein-protein interaction and provide solid evidence that the SH3 domain of BIN1 can establish an unexpectedly high number of interactions with at least a hundred cellular proteins, among which the authors underline the presence of other proteins known to be causative of skeletal muscle diseases and not known to interact with BIN1. This represents an unexpected and interesting finding relevant to better define the network of interactions established among different proteins that, if altered, can lead to muscle disease. An interesting contribution is also the detailed identification of the specific sites, namely the Proline-Rich Motifs (PRMs) that in the interacting proteins mediate binding to the BIN1 SH3 domain. Less convincing, or too preliminary in my opinion, are the data supporting BIN1 co-localization with PRC1. Indeed, the affinity of PRC1 is significantly lower than that of DNM2, an established BIN1 interacting protein. Thus, this does not provide compelling evidence to support PRC1 as a significant interactor of BIN1. Similarly, the localization data appears somewhat preliminary to substantiate a role of BIN1 in mitotic processes. These findings may necessitate additional experimental work to be more convincing.

Comments on revision:
I acknowledge the significant changes made by the authors in the revised manuscript. However, I remain puzzled by the data concerning the interaction between BIN1 and PRC1. While I agree with the authors that even weak interactions among proteins can be significant, I am hesitant to accept a priori that the lack of clear evidence of colocalization between proteins can be justified solely by their low affinity.

Moreover, the possibility that other mitotic proteins may be potential partners of BIN1 does not inherently support an interaction between BIN1 and PRC1. I suggest that the authors present the interaction with PRC1 as a potential event and emphasize that further studies are needed to definitively establish it.

Reviewer #2 (Public Review):

Original review:
Summary:
In this paper, Zambo and coworkers use a powerful technique, called native holdup, to measure the affinity of the SH3 domain of BIN1 for cellular partners. Using this assay, they combine data using cellular proteins and proline-containing fragments in these proteins to identify 97 distinct direct binding partners of BIN1. They also compare the binding interactome of the BIN1 SH3 domain to the interactome of several other SH3 domains, showing varying levels of promiscuity among SH3 domains. The authors then use pathway analysis of BIN1 binding partners to show that BIN1 may be involved in mitosis. Finally, the authors examine the impact of clinically relevant mutations of the BIN1 SH3 domain on the cellular interactome. The authors were able to compare the interactome of several different SH3 domains and provide novel insight into the cellular function of BIN1. Generally, the data supports the conclusions, although the reliance on one technique and the low number of replicates in each experiment is a weakness of the study.

Strengths:
The major strength of this paper is the use of holdup and native holdup assays to measure the affinity of SH3 domains to cellular partners. The use of both assays using cell-derived proteins and peptides derived from identified binding partners allows the authors to better identify direct binding partners. This assay has some complexity but does hold the possibility of being used to measure the affinity of the cellular interactome of other proteins and protein domains. Beyond the utility of the technique, this study also provides significant insight into the cellular function of BIN1. The authors have strong evidence that BIN1 might have an undiscovered function in cellular mitosis, which potentially highlights BIN1 as a drug target. Finally, the study provides outstanding data on the cellular binding properties and partners of seven distinct SH3 domains, showing surprising differences in the promiscuity of these proteins.

Weaknesses:
There are three major weaknesses of the study. First, the authors rely completely on a single technique to measure the affinity of the cellular interactome. The native holdup is a relatively new technique that is powerful yet relatively unproven. However, it appears to have the capacity to measure the relative affinity of proteins. Second, the authors appear to use a relatively small number of replicates for the holdup assays. There is no information in the legends about the number of replicates but the materials and methods suggest the native holdup data is from a single experimental replicate with multiple technical replicates. Finally, the authors' data using cellular proteins and fragments show that the affinity of the whole proteins is 5-20 fold lower than individual proline-containing fragments. The authors state that this difference suggests that there is cooperativity between different proline-rich sites of the binding partners of BIN1, yet BIN1 only has one SH3 domain. It is unclear what the molecular mechanism of the cooperative interaction would be exactly since there would be only one SH3 domain to bind the partner. An alternative interpretation would be that the BIN 1 SH3 domain requires sequences outside of the short proline-rich regions for high-affinity interactions with cellular partners, a hypothesis that is supported by other studies.

Comments on revision:
I thank the authors for their thoughtful response. I have additional comments.

I appreciate that this is not a techniques paper and that the authors have done more detailed work in a separate publication. It would be helpful to readers not familiar with this new method to more fully describe this technique in this manuscript.

I also thank the authors for their description of why they performed only 1 biological replicate of the experiment. However, I still believe that multiple biological replicates will provide more rigorous and reproducible data. The data the authors provide actually argues for the inclusion of more biological replicates. They state they performed 2 separate nHU replicates using different mass spectrometers. It is unclear if this data uses the same lysates and protein preparations, but by the data, the two methods detected a total of 207 distinct binding partners. Only 29 of these were significant binders in both replicates and only 90 were detected binders in both replicates. 117 binding partners were found in only one replicate suggesting a significant differences between replicates. Different batches of SH3 domains can have different activities and different replicates of cell lysates can vary, even when made from the same cell line. Thus, there can still be significant differences between replicates in this method. I appreciate the difficulty of performing and analyzing multiple biological replicates, but it is the most rigorous way to identify potential cellular partners.

I also thank the author for including the mechanistic discussion about the differences between peptides and whole proteins. There is literature showing that regions outside of the short PxxP regions drive binding to SH3 domains, especially for the GRB2 family of adaptor proteins.

Author response:

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

Reviewer #1:

We are grateful for the overall positive feedback from the reviewer.

We agree with the reviewer that our data showing cellular co-localization between PRC1 and BIN1 requires further investigation in future studies, however, we are confident that in the current form, our manuscript already presents multiple evidences for the role of BIN1 in mitotic processes. We would like to emphasize that PRC1 is not the sole BIN1 partner that connects it to mitotic processes, but it is only one out of more than a dozen that we identified in our study. Furthermore, the mitotic connection with BIN1 is not absolutely novel as BIN1 levels are mildly fluctuating during the cell cycle, similar to other proteins involved in the regulation of the cell cycle (Santos et al., 2015) and because DNM2 is also a well-accepted actor during mitosis (Thompson et al., 2002).

The less marked co-localization between BIN1 and PRC1 compared to the strong co-localization between BIN1 and DNM2 can be a consequence of their weaker affinity and their partial binding. Yet, this does not necessarily imply that stronger interactions have more biological significance. For example, weaker affinities can be compensated by local concentrations to achieve an even higher degree of cellular complexes than of strongly binding interactions that are separated within the cell. Furthermore, even the degree of complex formation cannot be used intuitively to estimate the biological significance of a complex because complexes can trigger very important biological processes even at very low abundances, e.g. by catalyzing enzymatic reactions. Deciding what is and what is not “biologically significant” among the identified interactions remains to be answered in the future, once we are able to overview complex biological processes in a holistic manner.

In the revised version, we implemented minor changes to further clarify the raised points.

Reviewer #2:

We thank the reviewer for the careful assessment and we are pleased to see the positive enthusiasm regarding our affinity interactomic strategy.

The reviewer points out that affinities were only measured with a single technique, which is relatively unproven. While it is true that our work uses two techniques building on the same holdup concept, we rather believe that this approach is well-proven. The original holdup method was described almost 20 years ago and since then, it has been used in more than 10 publications for quantitative interactomics. Over the years, at least five distinct generations of the assay were developed, all building on the expertise of the preceding one. In the past, we extensively proved that the resulting affinities show excellent agreement with affinities measured with other methods, such as fluorescence polarization, isothermal titration calorimetry, or surface plasmon resonance (for example in Vincentelli et al. Nat. Meth. 2015; Gogl et al. 2020 Structure; Gogl et al. 2022 Nat.Com.). However, it is true that the most recent variation of this method family, called native holdup, is a fairly new approach published just a bit more than a year ago and this is only the third work that utilizes this method. Yet, in our original work describing the method, we demonstrated good agreement with the results of previous holdup experiments, as well as with orthogonal affinity measurements (Zambo et al. 2022).

Importantly, the reviewer raises concerns regarding the number of replicates used in our study, as well as the reliability of our methodology. We are glad for such a comment as it allows us to explain our motives behind experimental design which is most often left out from scientific works to save space and keep focus on results. The reason why we use technical replicates instead of the typical biological replicates lies in the nature of the holdup assay. In a typical interactomic assay, such as immunoprecipitation, a lot of variables can perturb the outcome of the measurement, such as bait immobilization, or captured prey leakage during washing steps. The output of such an experiment is a list of statistically significant partners and to minimize these variabilities, biological replicates are used. In the case of a native holdup approach, a panel of an equal amount of resins, all saturated with different baits or controls, is mixed with an equal amount of cell extract, taken from a single tube, and after a brief incubation, the supernatant of this mixture is analyzed. The output of such an experiment is a list of relative concentrations of prey and to maximize its accuracy, we use technical replicates. Using an ideal analytical method, such as fluorescence, it is not necessary to use technical replicates to reach accurate results. For example, the general accuracy of a holdup experiment coupled with a robust analytical approach can be seen clearly in our fragmentomic holdup data shown in Figure 7C where mutant domains that do not have any impact on the interactome show extreme agreement in affinities. Unfortunately, mass spectrometry is less accurate as an analytical method, hence we use technical triplicates to compensate for this. Finally, in the case of BIN1, an independent nHU measurement was also performed using a less capable mass spectrometer. Not counting the 117 detected partners of BIN1 that were only detected in only one of these proteomic measurements, 29 partners were identified as common significant partners in both of these measurements showing nearly identical affinities with a mean standard deviation between measured pKapp values of 0.18, meaning that the obtained dissociation constants are within a <2.5-fold range with >95% probability. There were also 61 BIN1 partners that were detected in both proteomic measurements but were only identified as a significant interaction partner in one of these experiments. Yet many of them show binding in both assays, albeit were found to be not significant in one of these assays. For example, CDC20 shows 66% depletion in one assay (significant binding) while it shows 54% depletion in the other (not significant binding), or CKAP2 shows 58% depletion in one assay (significant binding) while it shows 41% depletion in the other (not significant binding). We hope that these examples show that statistical significance in nHU experiments rather signifies how certain we are in a particular affinity measurement and not the accuracy of the affinity measurement itself. While there are true discrepancies between some of the affinity measurements between these experiments, that would be possible to clarify with more experimental replicates, the raw data presented in our work clearly demonstrate the strength and robustness of a fully quantitative interactomic assay.

In the revised version, we clarified the number of replicates in the text, in the figure legends, and included some of this discussion in the method section.

The reviewer had some very useful comments regarding affinity differences between short fragments and full-length proteins. In his comment, he possibly made a typo as we find that fulllength proteins typically interact with higher affinities compared to short PxxP motif fragments in isolation and not weaker. The reviewer also comments that we explain this difference with cooperativity. In a previous preprint version, which the reviewer may have seen, this was indeed the case, but since we realized that we did not have sufficient evidence supporting this model, therefore we did not discuss this in detail in the last version submitted to eLife. To clarify this, we included more discussion about the observed differences in the affinities between fragments and full-length proteins, but since we have limited data to make solid conclusions, we do not go into details about underlying models.

Instead of cooperativity, the reviewer suggests that the observed differences may originate from additional residues that were not included in our peptides. Indeed, many similar experiments fail because of suboptimal peptide library design. Our peptide library was constructed as 15-mer, xxxxxxPxxPxxxxx motifs and we do not see a strong contribution of residues at the far end of these peptides. Specificity logo reconstructions are expected to identify all key residues that participate in SH3 domain binding, and based on this, all key residues of the identified motifs can be included in shorter 10-mer, xxxPxxPxxx motifs. Therefore, it is unlikely that residues outside our peptide regions will greatly contribute to the site-specific interactions of SH3 domains. It is however possible that other sites, that are sequentially far away from the studied PxxP motifs, are also capable of binding to SH3 through a different surface, but in light of the small size of an isolated SH3 domain, we believe it is very unlikely. It is also possible that BIN1 could also interact with other types of SH3 binding motifs that were not included in our peptide library. We think a more likely explanation is some sort of cooperativity. Cooperativity, or rather synergism between different sites can be easily explained in typical situations, such as in the case of a bimolecular interaction that is mediated by two independent sites. In such an event, once one site is bound, the second binding event will likely also occur because of the high effective local concentration of the binding sites. However, cooperativity can also form in atypical conditions and a molecular explanation for these events is rather elusive. As BIN1 contains a single SH3 domain, its binding to targets containing more binding sites can be challenging to interpret. If these sites are part of a greater Pro-rich region, such as in the case of DNM2, it is possible that the entire region adopts a fuzzy, malleable, yet PPII-like helical conformation. Once the SH3 domain is recruited to this helical region, it can freely trans-locate within this region via lateral diffusion and it will pause on optimal PxxP motifs. As an alternative to this sliding mechanism, a diffusion-limited cooperative binding can also occur. If the two motifs are not part of the same Pro-rich region, but are relatively close in space, such as in the case of ITCH or PRC1, once a BIN1 molecule dissociates from one site, it has a higher chance to rebind to the second site due to higher local concentrations. Such an event can more likely occur if a transient, but relatively stable encounter complex exists between the two molecules, from which complex formation can occur at both sites (A+B↔AB; AB↔ABsite1; AB*↔ABsite2). However, this large effective local concentration in this encounter complex is only temporary because diffusion rapidly diminishes it, although weak electrostatic interactions can increase the lifetime of such encounter complexes. In contrast, the large effective local concentration in conventional multivalent binding is time-independent and only determined by the geometry of the complex. Finally, it may also occur that our empirical bait concentration estimation for immobilized biotinylated proteins is less accurate than the concentration estimation of peptide baits because we approximate this value based on peptide baits. For this technical reason, which was discussed in detail in the original paper describing the nHU approach, we are carefully using apparent affinities for nHU experiments. Nevertheless, even without accurate bait concentrations, our nHU experiment provides precise relative affinities and, thus partner ranking. Either of the mechanisms underlying the interactions we study would be difficult to further explore experimentally, especially at the proteomic level.