Single Domain Antibody Inhibitors Target the Coiled Coil Arms of the Bacillus subtilis SMC complex

  1. Ophélie Gosselin
  2. Michael Taschner
  3. Lea M Huber-Hürlimann
  4. Markus A Seeger
  5. Stephan Gruber

  • Curated by eLife

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    eLife Assessment

    This important study introduces an innovative synthetic nanobody approach to probe the function of the bacterial SMC complex. The authors provide convincing evidence that these nanobodies target the coiled-coil region of the SMC subunit and demonstrate that this region is critical for SMC function in vivo. Overall, the work is significant for the fields of genome organization, SMC protein biology, synthetic biology, and bacterial cell biology.

    [Editors' note: this paper was reviewed by Review Commons.]

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Abstract

Synthetic nanobodies—also called sybodies—have proven valuable for stabilizing conformations of purified proteins, advancing structural and functional studies for example of transmembrane protein complexes. However, their utility in modulating protein function in living cells has remained less well explored. Structural Maintenance of Chromosomes (SMC) complexes facilitate chromosome organization by DNA loop extrusion, a fundamental process in all domains of life. In this study, we target the bacterial SMC complex, Smc-ScpAB, in Bacillus subtilis with synthetic nanobodies, with the aim to identify key functional regions of the protein complex in a largely unbiased manner. We first isolate sybodies that specifically bind purified Smc-ScpAB and then express them in B. subtilis to select binders capable of disrupting Smc-ScpAB function, leading to chromosome segregation defects and cell death. Mapping and biochemical characterization show that the fourteen disruptive sybodies belong to one of three library designs, target the Smc subunit near the same coiled coil arm interface and modulate its ATPase activity in two principal ways, highlighting the mid-region of the Smc coiled coil as critical feature of the DNA folding process. These findings underscore the potential of sybodies—and, by extension, designed binders—as versatile tools for probing dynamic protein function in living cells, with potentially broad applications in cell and synthetic biology.

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  1. Version published to 10.7554/elife.111131.1 on eLife
  2. Version published to 10.7554/elife.111131 on eLife
  3. eLife

    eLife Assessment

    This important study introduces an innovative synthetic nanobody approach to probe the function of the bacterial SMC complex. The authors provide convincing evidence that these nanobodies target the coiled-coil region of the SMC subunit and demonstrate that this region is critical for SMC function in vivo. Overall, the work is significant for the fields of genome organization, SMC protein biology, synthetic biology, and bacterial cell biology.

    [Editors' note: this paper was reviewed by Review Commons.]

  4. eLife

    Reviewer #1 (Public review):

    Summary:

    Gosselin et al., develop a method to target protein activity using synthetic single-domain nanobodies (sybodies). They screen a library of sybodies using ribosome/ phage display generated against bacillus Smc-ScpAB complex. Specifically, they use an ATP hydrolysis deficient mutant of SMC so as to identify sybodies that will potentially disrupt Smc-ScpAB activity. They next screen their library in vivo, using growth defects in rich media as a read-out for Smc activity perturbation. They identify 14 sybodies that mirror smc deletion phenotype including defective growth in fast-growth conditions, as well as chromosome segregation defects. The authors use a clever approach by making chimeras between bacillus and S. pnuemoniae Smc to narrow-down to specific regions within the bacillus Smc coiled-coil that are likely targets of the sybodies. Using ATPase assays, they find that the sybodies either impede DNA-stimulated ATP hydrolysis or hyperactivate ATP hydrolysis (even in the absence of DNA). The authors propose that the sybodies may likely be locking Smc-ScpAB in the "closed" or "open" state via interaction with the specific coiled-coil region on Smc. I have a few comments that the authors should consider:

    Major comments:

    (1) Lack of direct in vitro binding measurements:
    The authors do not provide measurements of sybody affinities, binding/ unbinding kinetics, stoichiometries with respect to Smc-ScpAB. Additionally, do the sybodies preferentially interact with Smc in ATP/ DNA-bound state? And do the sybodies affect the interaction of ScpAB with SMC?
    It is understandable that such measurements for 14 sybodies is challenging, and not essential for this study. Nonetheless, it is informative to have biochemical characterization of sybody interaction with the Smc-ScpAB complex for at least 1-2 candidate sybodies described here.

    (2) Many modes of sybody binding to Smc are plausible
    The authors provide an elaborate discussion of sybodies locking the Smc-ScpAB complex in open/ closed states. However, in the absence of structural support, the mechanistic inferences may need to be tempered. For example, is it also not possible for the sybodies to bind the inner interface of the coiled-coil, resulting in steric hinderance to coiled-coil interactions. It is also possible that sybody interaction disrupts ScpAB interaction (as data ruling this possibility out has not been provided). Thus, other potential mechanisms would be worth considering/ discussing. In this direction, did AlphaFold reveal any potential insights into putative binding locations?

    (3) Sybody expression in vivo
    Have the authors estimated sybody expression in vivo? Are they all expressed to similar levels?

    (4) Sybodies should phenocopy ATP hydrolysis mutant of Smc
    The sybodies were screened against an ATP hydrolysis deficient mutant of Smc, with the rationale that these sybodies would interfere this step of the Smc duty cycle. Does the expression of the sybodies in vivo phenocopy the ATP hydrolysis deficient mutant of Smc? Could the authors consider any phenotypic read-outs that can indicate whether the sybody action results in an smc-null effect or specifically an ATP hydrolysis deficient effect?

    Significance:

    Overall, this is an impressive study that uses an elegant strategy to find inhibitors of protein activity in vivo. The manuscript is clearly written and the experiments are logical and well-designed. The findings from the study will be significant to the broad field of genome biology, synthetic biology and also SMC biology. Specifically, the coiled coil domain of SMC proteins have been proposed to be of high functional value. The authors have elegantly identified key coiled-coil regions that may be important for function, and parallelly exhibited potential of the use of synthetic sybody/designed binders for inhibition of protein activity.

  5. eLife

    Reviewer #2 (Public review):

    Summary:

    Structural Maintenance of Chromosome proteins (SMCs), a family of proteins found in almost all organisms, are organizers of DNA. They accomplish this by a process known as loop extrusion, wherein double-stranded DNA is actively reeled in and extruded into loops. Although SMCs are known to have several DNA binding regions, the exact mechanism by which they facilitate loop extrusion is not understood but is believed to entail large conformational changes. There are currently several models for loop extrusion, including one wherein the coiled coil (CC) arms open, but there is a lack of insightful experimentation and analysis to confirm any of these models. The work presented aims to provide much-needed new tools to investigate these questions: conformation-selective sybodies (synthetic nanobodies) that are likely to alter the CC opening and closing reactions.

    The authors produced, isolated, and expressed sybodies that specifically bound to Bacillus subtilis Smc-ScpAB. Using chimeric Smc constructs, where the coiled coils were partly replaced with the corresponding sequences from Streptococcus pneumoniae, the authors revealed that the isolated sybodies all targeted the same 4N CC element of the Smc arms. This region is likely disrupted by the sybodies either by stopping the arms from opening (correctly) or forcing them to stay open (enough). Disrupting these functional elements is suggested to cause the Smc-dependent chromosome organization lethal phenotype, implying that arm opening and closing is a key regulatory feature of bacterial Smc-ScpAB.

    Significance:

    The authors present a new method for trapping bacterial Smc's in certain conformations using synthetic antibodies. Using these antibodies, they have pinpointed the (previously suggested) 4N region of the coiled coils as an essential site for the opening and closing of the Smc coiled coil arms and that hindering these reactions blocks Smc-driven chromosomal organization. The work has important implications for how we might elucidate the mechanism of DNA loop extrusion by SMC complexes.

  6. eLife

    Reviewer #3 (Public review):

    Summary:

    Gosselin et al. use the sybody technology to study effects of in vivo inhibition of the Bacillus subtilis SMC complex. Smc proteins are central DNA binding elements of several complexes that are vital for chromosome dynamics in almost all organisms. Sybodies are selected from three different libraries of the single domain antibodies, using the "transition state" mutant Smc. They identify 14 such mutant sybodies that are lethal when expressed in vivo, because they prevent proper function of Smc. The authors present evidence suggesting that all obtained sybodies bind to a coiled-coil region close to the Smc "neck", and thereby interfere with the Smc activity cycle, as evidenced by defective ATPase activity when Smc is bound to DNA.
    The study is well done and presented and shows that the strategy is very potent in finding a means to quickly turn off a protein's function in vivo, much quicker than depleting the protein.

    The authors also draw conclusions on the molecular mode of action of the SMC complex. The provide a number of suggestive experiments, but in my view mostly indirect evidence for such mechanism.

    My main criticism is that the authors have used a single - and catalytically trapped form of SMC. They speculate why they only obtain sybodies from one library, and then only identify sybodies that bind to a rather small part of the large Smc protein. While the approach is definitely valuable, it is biassed towards sybodies that bind to Smc in a quite special way, it seems. Using wild type Smc would be interesting, to make more robust statements about the action of sybodies potentially binding to different parts of Smc.

    Line 105: Alternatively, the other libraries did not produce good binders or these sybodies were 106 not stably expressed in B. subtilis. This could be tested using Western blotting - I am assuming sybody antibodies are commercially available. However, this test is not important for the overall study, it would just clarify a minor point.

    Fig. 2B: is odd to count Spo0J foci per cells, as it is clear from the images that several origins must be present within the fluorescent foci. I am fine with the "counting" method, as the images show there is a clear segregation defect when sybodies are expressed, I believe the authors should state, though, that this is not a replication block, but failure to segregate origins.

    Testing binding sites of sybodies to the SMC complex is done in an indirect manner, by using chimeric Smc constructs. I am surprised why the authors have not used in vitro crosslinking: the authors can purify Smc, and mass spectrometry analyses would identify sites where sybodies are crosslinked to Smc. Again, I am fine with the indirect method, but the authors make quite concrete statements on binding based on non-inhibition of chimeric Smc; I can see alternative explanations why a chimera may not be targeted.

    Smc-disrupting sybodies affect the ATPase activity in one of two ways. Again, rather indirect experiments. This leads to the point Revealing Smc arm dynamics through synthetic binders in the discussion. The authors are quite careful in stating that their experiments are suggestive for a certain mode of action of Smc, which is warranted.

    In line 245, they state More broadly, the study demonstrates how synthetic binders can trap, stabilize, or block transient conformations of active chromatin-associated machines, providing a powerful means to probe their mechanisms in living cells. This is off course a possible scenario for the use of sybodies, but the study does not really trap Smc in a transient conformation, at least this is not clearly shown.

    Overall, it is an interesting study, with a well-presented novel technology, and a limited gain of knowledge on SMC proteins.

    Significance:

    The work describes the gaining and use of single-binder antibodies (sybodies) to interfere with the function of proteins in bacteria. Using this technology for the SMC complex, the authors demonstrate that they can obtain a significant of binders that target a defined region is SMC and thereby interfere with the ATPase cycle.

    The study does not present a strong gain of knowledge of the mode of action of the SMC complex.

  7. eLife

    Author response:

    General Statements

    First, we would like to thank the editor at Review Commons for the efficient handling of our manuscript. We also apologize for our delayed response.

    We would like to thank all three reviewers for their careful evaluation of our work and their constructive feedback, which will provide a valuable basis for improving the figures and the text, as described below. We expect to be able to complete the revision following the plan described below quickly.

    We would like to note that the reviewer reports (Rev. #1 and Rev. #3) made us realize that the manuscript text was misleading on the following point. Although we used the purified ATP hydrolysis–deficient Smc protein for sybody isolation, this does not restrict the selection to a specific conformation. As described in detail in Vazquez-Nunez et al. (Figure 5), this mutant displays the ATP-engaged conformation only in a smaller fraction of complexes (~25% in the presence of ATP and DNA), consistent with prior in vivo observations reported by Diebold-Durand et al. (Figure 5). Rather than limiting the selection to a particular configuration, our aim was to reduce the prevalence of the predominant rod state in order to broaden the range of conformations represented during sybody selection. Consistent with this interpretation, only a small number of isolated sybodies show strong conformation-specific binding in the presence or absence of ATP/DNA, as observed by ELISA (now included in the manuscript). We will revise the manuscript text accordingly to clarify this point.

    Description of the planned revisions

    Reviewer #1 (Evidence, reproducibility and clarity):

    Gosselin et al., develop a method to target protein activity using synthetic single-domain nanobodies (sybodies). They screen a library of sybodies using ribosome/ phage display generated against bacillus Smc-ScpAB complex. Specifically, they use an ATP hydrolysis deficient mutant of SMC so as to identify sybodies that will potentially disrupt Smc-ScpAB activity. They next screen their library in vivo, using growth defects in rich media as a read-out for Smc activity perturbation. They identify 14 sybodies that mirror smc deletion phenotype including defective growth in fast-growth conditions, as well as chromosome segregation defects. The authors use a clever approach by making chimeras between bacillus and S. pnuemoniae Smc to narrow-down to specific regions within the bacillus Smc coiled-coil that are likely targets of the sybodies. Using ATPase assays, they find that the sybodies either impede DNA-stimulated ATP hydrolysis or hyperactivate ATP hydrolysis (even in the absence of DNA). The authors propose that the sybodies may likely be locking Smc-ScpAB in the "closed" or "open" state via interaction with the specific coiled-coil region on Smc. I have a few comments that the authors should consider:

    Major comments:

    (1) Lack of direct in vitro binding measurements:

    The authors do not provide measurements of sybody affinities, binding/ unbinding kinetics, stoichiometries with respect to Smc-ScpAB. Additionally, do the sybodies preferentially interact with Smc in ATP/ DNA-bound state? And, do the sybodies affect the interaction of ScpAB with SMC?

    It is understandable that such measurements for 14 sybodies is challenging, and not essential for this study. Nonetheless, it is informative to have biochemical characterization of sybody interaction with the Smc-ScpAB complex for at least 1-2 candidate sybodies described here.

    We agree with the reviewer that adding such data would be reassuring and that obtaining solid data using purified components is not easy even for a smaller selection of sybodies. We have data that show direct binding of Smc to sybodies by various methods including ELISA, pull-downs and by biophysical methods (GCI). Initially, we omitted these data from the manuscript as we are convinced that the mapping data obtained with chimeric SMC proteins is more definitive and relevant. During the revision we will incorporate the ELISA data showing direct binding and also indicating a lack of preference for a specific state of Smc.

    (2) Many modes of sybody binding to Smc are plausible

    The authors provide an elaborate discussion of sybodies locking the Smc-ScpAB complex in open/ closed states. However, in the absence of structural support, the mechanistic inferences may need to be tempered. For example, is it also not possible for the sybodies to bind the inner interface of the coiled-coil, resulting in steric hinderance to coiled-coil interactions. It is also possible that sybody interaction disrupts ScpAB interaction (as data ruling this possibility out has not been provided). Thus, other potential mechanisms would be worth considering/ discussing. In this direction, did AlphaFold reveal any potential insights into putative binding locations?

    We have attempted to map the binding by structure prediction, however, so far, even the latest versions of AlphaFold are not able to clearly delineate the binding interface. Indeed, many ways of binding are possible, including disruption of ScpAB interaction. However, since the main binding site is located on the SMC coiled coils, the later scenario would likely be an indirect consequence of altered coiled coil configuration, consistent with our current interpretation.

    (3) Sybody expression in vivo

    Have the authors estimated sybody expression in vivo? Are they all expressed to similar levels?

    We have tagged selected sybodies with gfp and performed live cell imaging. This showed that they are all roughly equally expressed and that they localize as foci in the cell presumably by binding to Smc complexes loaded onto the chromosome at ParB/parS sites. We will include this data in the revised version of the manuscript.

    (4) Sybodies should phenocopy ATP hydrolysis mutant of Smc

    The sybodies were screened against an ATP hydrolysis deficient mutant of Smc, with the rationale that these sybodies would interfere this step of the Smc duty cycle. Does the expression of the sybodies in vivo phenocopy the ATP hydrolysis deficient mutant of Smc? Could the authors consider any phenotypic read-outs that can indicate whether the sybody action results in an smc-null effect or specifically an ATP hydrolysis deficient effect?

    As eluded to above, we think that our selection gave rise to sybodies that bind various, possibly multiple Smc conformations. Consistent with this idea, the phenotypes are similar to null mutant rather than the ATP-hydrolysis defective EQ mutant, which display even more severe growth phenotypes. We will add the following notes to the text:

    “These conditions favour ATP-engaged particles alongside the typically predominant ATP-disengaged rod-shaped state (add Vazquez Nunez et al., 2021).”

    “ELISA data confirm that nearly all clones bind Smc-ScpAB; however, their binding shows little or no dependence on the presence of ATP or DNA.”

    Minor comments:

    (1) It was surprising that no sybodies were found that could target both bacillus and spneu Smc. For example, sybodies targeting the head regions of Smc that might work in a more universal manner. Could the authors comment on the coverage of the sybodies across the protein structure?

    It is rather common that sybodies (like antibodies and nanobodies) exhibit strong affinity differences between highly conserved proteins (> 90 % identity). The underlying reasons for such strong discrimination are i) location of less conserved residues primarily at the target protein surface and ii) the large interaction interface between sybody and target which offers multiple vulnerabilities for disturbance, in particular through bulky side chains resulting in steric clashes. Another frequently observed phenomenon is sybody binding to a dominant epitope, which also often applies to nanobodies and antibodies. A great example for this are the dominant epitopes on SARS-CoV-2 RBDs.

    (2) Growth curves (Fig. S3) show a large jump in recovery in growth under sybody induction conditions. Could the authors address this observation here and in the text?

    We suppose that this recovery represents suppressor mutants and/or (more likely) improved growth in the absence of functional Smc during nutrient limitation (see Gruber et al., 2013 and Wang et al., 2013). We will add this statement to the text.

    (3) L41- Sentence correction: Loop can be removed.

    Ah, yes, sorry for this confusing error. Thank you.

    (4) L525 - bsuSmc 'E' :extra E can be removed.

    To do. Thank you.

    (5) References need to be properly formatted.

    To do. Thank you.

    (6) The authors should add in figure legend for Fig 1i) details on representation of the purple region, and explain the grey strokes for orientation of the loop.

    To do.

    (7) How many cells were analysed in the cell biological assays? Legends should include these information.

    To Be Included.

    Reviewer #1 (Significance):

    Overall, this is an impressive study that uses an elegant strategy to find inhibitors of protein activity in vivo. The manuscript is clearly written and the experiments are logical and well-designed. The findings from the study will be significant to the broad field of genome biology, synthetic biology and also SMC biology. Specifically, the coiled coil domain of SMC proteins have been proposed to be of high functional value. The authors have elegantly identified key coiled-coil regions that may be important for function, and parallelly exhibited potential of the use of synthetic sybody/designed binders for inhibition of protein activity.

    Reviewer #2 (Evidence, reproducibility and clarity):

    Review: "Single Domain Antibody Inhibitors Target the Coiled Coil Arms of the Bacillus subtilis SMC complex" by Ophélie Gosselin et al, Review Commons RC-2025-03280 Structural Maintenance of Chromosome proteins (SMCs), a family of proteins found in almost all organisms, are organizers of DNA. They accomplish this by a process known as loop extrusion, wherein double-stranded DNA is actively reeled in and extruded into loops. Although SMCs are known to have several DNA binding regions, the exact mechanism by which they facilitate loop extrusion is not understood but is believed to entail large conformational changes. There are currently several models for loop extrusion, including one wherein the coiled coil (CC) arms open, but there is a lack of insightful experimentation and analysis to confirm any of these models. The work presented aims to provide much-needed new tools to investigate these questions: conformation-selective sybodies (synthetic nanobodies) that are likely to alter the CC opening and closing reactions.

    The authors produced, isolated, and expressed sybodies that specifically bound to Bacillus subtilis Smc-ScpAB. Using chimeric Smc constructs, where the coiled coils were partly replaced with the corresponding sequences from Streptococcus pneumoniae, the authors revealed that the isolated sybodies all targeted the same 4N CC element of the Smc arms. This region is likely disrupted by the sybodies either by stopping the arms from opening (correctly) or forcing them to stay open (enough). Disrupting these functional elements is suggested to cause the Smc-dependent chromosome organization lethal phenotype, implying that arm opening and closing is a key regulatory feature of bacterial Smc-ScpAB.

    In summary, the authors present a new method for trapping bacterial Smc's in certain conformations using synthetic antibodies. Using these antibodies, they have pinpointed the (previously suggested) 4N region of the coiled coils as an essential site for the opening and closing of the Smc coiled coil arms and that hindering these reactions blocks Smc-driven chromosomal organization. The work has important implications for how we might elucidate the mechanism of DNA loop extrusion by SMC complexes.

    Some specific comments:

    Line 75: "likely stabilizing otherwise rare intermediates of the conformational cycle." - sorry, why is that being concluded? Why not stabilizing longer-lived oncformations?

    We will clarify this statement!

    Line 89: Sorry, possibly our lack of understanding: why first ribosome and then phage display?

    Ribosome display offers to screen around 10^12 sybodies per selection round (technically unrestricted library size), while for phage display, the library size is restricted to around 10^9 sybodies due to the fact that production of a phage library requires transformation of the phagemid plasmid into E. coli, thereby introducing a diversity bottleneck. This is why the sybody platform starts off with ribosome display. It switches to phage display from round 2 onwards because the output of the initial round of ribosome display is around 10^6 sybodies, which can be easily transferred into the phage display format. Phage display is used to minimize selection biases. For more information, please consult the original sybody paper (PMID: 29792401).

    Line 100: Why was only lethality selected? Less severe phenotypes not clear enough?

    Yes, colony size is more difficult to score robustly, as the sizes of individual transformant colonies can vary quite widely. The number of isolated sybodies was at the limit of further analysis.

    Line 106: Could it be tested somehow if convex and concave library sybodies fold in Bs?

    We did not focus on the non-functional sybody candidates and only sybodies of the loop library turned out to cause functional consequences at the cellular level. Notably, we will include gfp-imaging showing that non-lethal sybodies are expressed to similar levels that toxic sybodies. Given the identical scaffold of concave and loop sybodies (they only differ in their CDR3 length), we expect that the concave sybodies fold in the cytoplasm of B. subtilis. For the convex sybodies exhibiting a different scaffold, this will be tested.

    Line 125: Could Pxyl be repressed by glucose?

    To our knowledge and experience, repression by glucose (catabolite repression) does not work well in this context in B. subtilis.

    Line 131: The SMC replacement strain is a cool experiment and removes a lot of doubts!

    Thank you! (we agree).

    Line 141: The mapping is good and looks reliable, but looks and feels like a tour de force? Of course, some cryo-EM would have been lovely (lines 228-229 understood, it has been tried!).

    Yes, we have made several attempts at structural biology. Unfortunately, Smc-ScpAB is not well suited for cryo-EM in our hands and crystallography with Smc fragments and sybodies did not yield well-diffracting crystals.

    Line 179: Mmmh. Do we not assume DNA binding on top of the dimerised heads to open the CC (clamp)?

    We will clarify the text here.

    Line 187: Having sybodies that presumably keep the CC together (closing) and some that do not allow them to come together correctly (opening) is really cool and probably important going forward.

    Thank you!

    Figure 1 Ai is not very colour-blind friendly.

    We are sorry for this oversight. We will try to make the color scheme more inclusive. Thank you for the notification.

    Optional: did the authors see any spontaneous mutations emerge that bypass the lethal phenotype of sybody expression?

    No, we did not observe spontaneous mutations suppressing the phenotype, possibly due to the limited number of cell generations observed. We tried to avoid suppressors by limiting growth, but this may indeed be a good future approach for further fine map the binding sites and to obtain insights into the mechanism of inhibition.

    Optional: we think it would be nice to try some biochemical experiment with BMOE/cysteine-crosslinked B. subtilis Smc in the mid-region (4N or next to it) of the Smc coiled coils to try to further strengthen the story. Some of the authors are experts in this technique and strains might already exist?

    We have indeed tried to study the impact of sybody binding on Smc conformation by cysteine cross-linking. However, we were not convinced by the results and thus prefer not to draw any conclusions from them. We will add a corresponding note to the text.

    Reviewer #2 (Significance):

    The authors present a new method for trapping bacterial Smc's in certain conformations using synthetic antibodies. Using these antibodies, they have pinpointed the (previously suggested) 4N region of the coiled coils as an essential site for the opening and closing of the Smc coiled coil arms and that hindering these reactions blocks Smc-driven chromosomal organization. The work has important implications for how we might elucidate the mechanism of DNA loop extrusion by SMC complexes.

    Thank you!

    Reviewer #3 (Evidence, reproducibility and clarity):

    Gosselin et al. use the sybody technology to study effects of in vivo inhibition oft he Bacillus subtilis SMC complex. Smc proteins are central DNA binding elements of several complexes that are vital for chromosome dynamics in almost all organisms. Sybodies are selected from three different libraries of the single domain antibodies, using the „transition state" mutant Smc. They identify 14 such mutant sybodies that are lethal when expressed in vivo, because they prevent proper function of Smc. The authors present evidence suggesting that all obtained sybodies bind to a coiled-coil region close to the Smc „neck", and thereby interfere with the Smc activity cycle, as evidenced by defective ATPase activity when Smc is bound to DNA.

    The study is well done and presented and shows that the strategy is very potent in finding a means to quickly turn off a protein's function in vivo, much quicker than depleting the protein.

    The authors also draw conclusions on the molecular mode of action of the SMC complex. The provide a number of suggestive experiments, but in my view mostly indirect evidence for such mechanism.

    My main criticism ist hat the authors have used a single - and catalytically trapped form of SMC. They speculate why they only obtain sybodies from one library, and then only idenfity sybodies that bind to a rather small part oft he large Smc protein. While the approach is definitely valuable, it is biassed towards sybodies that bind to Smc in a quite special way, it seems. Using wild type Smc would be interesting, to make more robust statements about the action of sybodies potentially binding to different parts of Smc.

    As explained above, we are quite confident the Smc ATPase mutation did not bias the selection in an obvious way. The surprising bias towards coiled coil binding sites has likely other explanations, as they likely form a preferred epitope recognized by sybodies.

    Line 105: Alternatively, the other libraries did not produce good binders or these sybodies were 106 not stably expressed in B. subtilis. This could be tested using Western blotting - I am assuming sybody antibodies are commercially available. However, this test is not important for the overall study, it would just clarify a minor point.

    While there are antibody fragments available to augment the size of sybodies (PMID: 40108246), these recognize 3D-epitopes and are thus not suited for Western blotting. We did not follow up on the negative results much, but would like to point out again that there are several biases that likely emerge for the same reason (bias to library, bias to coiled coil binding site). If correct, then likely few other sybodies are effectively lethal in B. subtilis, with the exception of the ones isolated and characterized. We have added this notion to the manuscript. We have also tested the expression of non-lethal sybodies by gfp-tagging and imaging. These results will be included in the revision.

    Fig. 2B: is is odd to count Spo0J foci per cells, as it is clear from the images that several origins must be present within the fluorescent foci. I am fine with the „counting" method, as the images show there is a clear segregation defect when sybodies are expressed, I believe the authors should state, though, that this is not a replication block, but failure to segregate origins.

    We agree that this is an important point and will add a corresponding comment to the text.

    Testing binding sites of sybodies tot he SMC complex is done in an indirect manner, by using chimeric Smc constructs. I am surprised why the authors have not used in vitro crosslinking: the authors can purify Smc, and mass spectrometry analyses would identify sites where sybodies are crosslinked to Smc. Again, I am fine with the indirect method, but the authors make quite concrete statements on binding based on non-inhibition of chimeric Smc; I can see alternative explanations why a chimera may not be targeted.

    We have made several attempts of testing direct binding with mixed outcomes and decided to not include those results in the light of the stronger and more relevant in vivo mapping. However, we will add ELISA results and briefly discuss grating coupled interferometry (GCI) data and pull-downs.

    Smc-disrupting sybodies affect the ATPase activity in one of two ways. Again, rather indirect experiments. This leads to the point Revealing Smc arm dynamics through synthetic binders in the discussion. The authors are quite careful in stating that their experiments are suggestive for a certain mode of action of Smc, which is warranted.

    In line 245, they state More broadly, the study demonstrates how synthetic binders can trap, stabilize, or block transient conformations of active chromatin-associated machines, providing a powerful means to probe their mechanisms in living cells. This is off course a possible scenario for the use of sybodies, but the study does not really trap Smc in a transient conformation, at least this is not clearly shown.

    We agree and will carefully rephrase this statement. Thank you.

    Overall, it is an interesting study, with a well-presented novel technology, and a limited gain of knowledge on SMC proteins.

    We respectfully disagree with the last point, since our unique results highlight the importance of the Smc coiled coils, which are otherwise largely neglected in the SMC literature, likely (at least in part) due the mild effect of single point mutations on coiled coil dynamics.

    Reviewer #3 (Significance):

    The work describes the gaining and use of single-binder antibodies (sybodies) to interfere with the function of proteins in bacteria. Using this technology for the SMC complex, the authors demonstrate that they can obtain a significant of binders that target a defined region is SMC and thereby interfere with the ATPase cycle.

    The study does not present a strong gain of knowledge of the mode of action of the SMC complex.

    As pointed out above, we respectfully disagree with this assertion.

    Description of analyses that authors prefer not to carry out

    As pointed out above, there are a few minor points that we prefer not to experimentally address. In particular, we do not consider it as necessary to determine the expression levels of sybodies which were non-inhibitory. We also wish to note that we attempted to obtain structural additional biochemical data and to that end performed cryo-EM, crystallography and cysteine cross-linking experiments. Unfortunately, we did not obtain sybody complex structures and the cross-linking data were unfortunately not conclusive. We also wish to note that the first author has finished her PhD and left the lab, which limits our capacity to add additional experiments. However, as the reviewers also pointed out, the main conclusions are well supported by the data already.

  8. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    Manuscript number: RC-2025-03280

    Corresponding author(s): Stephan Gruber

    1. General Statements [optional]

    First, we would like to thank the editor at Review Commons for the efficient handling of our manuscript. We also apologize for our delayed response.

    We are grateful to all three reviewers for their careful evaluation of our work and for their constructive feedback, which will provide a valuable basis for improving the figures and the text, as described below. We expect to be able to complete the revision following the plan described below quickly.

    We note that the reviewer reports (Rev. #1 and Rev. #3) made us realize that the manuscript text was misleading on the following point. Although we used the purified ATP hydrolysis–deficient Smc protein for sybody isolation, this does not restrict the selection to a specific conformation. As described in detail in Vazquez-Nunez et al. (Figure 5), this mutant displays the ATP-engaged conformation only in a smaller fraction of complexes (~25% in the presence of ATP and DNA), consistent with prior in vivo observations reported by Diebold-Durand et al. (Figure 5). Rather than limiting the selection to a particular configuration, our aim was to reduce the prevalence of the predominant rod state in order to broaden the range of conformations represented during sybody selection. Consistent with this interpretation, only a small number of isolated sybodies show strong conformation-specific binding in the presence or absence of ATP/DNA, as observed by ELISA (now included in the manuscript). We will revise the manuscript text accordingly to clarify this point.

    2. Description of the planned revisions

    Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees.

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    Gosselin et al., develop a method to target protein activity using synthetic single-domain nanobodies (sybodies). They screen a library of sybodies using ribosome/ phage display generated against bacillus Smc-ScpAB complex. Specifically, they use an ATP hydrolysis deficient mutant of SMC so as to identify sybodies that will potentially disrupt Smc-ScpAB activity. They next screen their library in vivo, using growth defects in rich media as a read-out for Smc activity perturbation. They identify 14 sybodies that mirror smc deletion phenotype including defective growth in fast-growth conditions, as well as chromosome segregation defects. The authors use a clever approach by making chimeras between bacillus and S. pnuemoniae Smc to narrow-down to specific regions within the bacillus Smc coiled-coil that are likely targets of the sybodies. Using ATPase assays, they find that the sybodies either impede DNA-stimulated ATP hydrolysis or hyperactivate ATP hydrolysis (even in the absence of DNA). The authors propose that the sybodies may likely be locking Smc-ScpAB in the "closed" or "open" state via interaction with the specific coiled-coil region on Smc. I have a few comments that the authors should consider:

    Major comments:

    1. Lack of direct in vitro binding measurements: The authors do not provide measurements of sybody affinities, binding/ unbinding kinetics, stoichiometries with respect to Smc-ScpAB. Additionally, do the sybodies preferentially interact with Smc in ATP/ DNA-bound state? And, do the sybodies affect the interaction of ScpAB with SMC? It is understandable that such measurements for 14 sybodies is challenging, and not essential for this study. Nonetheless, it is informative to have biochemical characterization of sybody interaction with the Smc-ScpAB complex for at least 1-2 candidate sybodies described here.

    We agree with the reviewer that adding such data would be reassuring and that obtaining solid data using purified components is not easy even for a smaller selection of sybodies. We have data that show direct binding of Smc to sybodies by various methods including ELISA, pull-downs and by biophysical methods (GCI). Initially, we omitted these data from the manuscript as we are convinced that the mapping data obtained with chimeric SMC proteins is more definitive and relevant. During the revision we will incorporate the ELISA data showing direct binding and also indicating a lack of preference for a specific state of Smc.

    Many modes of sybody binding to Smc are plausible The authors provide an elaborate discussion of sybodies locking the Smc-ScpAB complex in open/ closed states. However, in the absence of structural support, the mechanistic inferences may need to be tempered. For example, is it also not possible for the sybodies to bind the inner interface of the coiled-coil, resulting in steric hinderance to coiled-coil interactions. It is also possible that sybody interaction disrupts ScpAB interaction (as data ruling this possibility out has not been provided). Thus, other potential mechanisms would be worth considering/ discussing. In this direction, did AlphaFold reveal any potential insights into putative binding locations?

    We have attempted to map the binding by structure prediction, however, so far, even the latest versions of AlphaFold are not able to clearly delineate the binding interface. Indeed, many ways of binding are possible, including disruption of ScpAB interaction. However, since the main binding site is located on the SMC coiled coils, the later scenario would likely be an indirect consequence of altered coiled coil configuration, consistent with our current interpretation.

    1. Sybody expression in vivo Have the authors estimated sybody expression in vivo? Are they all expressed to similar levels?

    We have tagged selected sybodies with gfp and performed live cell imaging. This showed that they are all roughly equally expressed and that they localize as foci in the cell presumably by binding to Smc complexes loaded onto the chromosome at ParB/parS sites. We will include this data in the revised version of the manuscript.

    1. Sybodies should phenocopy ATP hydrolysis mutant of Smc The sybodies were screened against an ATP hydrolysis deficient mutant of Smc, with the rationale that these sybodies would interfere this step of the Smc duty cycle. Does the expression of the sybodies in vivo phenocopy the ATP hydrolysis deficient mutant of Smc? Could the authors consider any phenotypic read-outs that can indicate whether the sybody action results in an smc-null effect or specifically an ATP hydrolysis deficient effect?

    As eluded to above, we think that our selection gave rise to sybodies that bind various, possibly multiple Smc conformations. Consistent with this idea, the phenotypes are similar to null mutant rather than the ATP-hydrolysis defective EQ mutant, which display even more severe growth phenotypes. We will add the following notes to the text:

    “These conditions favour ATP-engaged particles alongside the typically predominant ATP-disengaged rod-shaped state (add Vazquez Nunez et al., 2021).”

    “ELISA data confirm that nearly all clones bind Smc-ScpAB; however, their binding shows little or no dependence on the presence of ATP or DNA.”

    Minor comments:

    1. It was surprising that no sybodies were found that could target both bacillus and spneu Smc. For example, sybodies targeting the head regions of Smc that might work in a more universal manner. Could the authors comment on the coverage of the sybodies across the protein structure?

    It is rather common that sybodies (like antibodies and nanobodies) exhibit strong affinity differences between highly conserved proteins (> 90 % identity). The underlying reasons for such strong discrimination are i) location of less conserved residues primarily at the target protein surface and ii) the large interaction interface between sybody and target which offers multiple vulnerabilities for disturbance, in particular through bulky side chains resulting in steric clashes. Another frequently observed phenomenon is sybody binding to a dominant epitope, which also often applies to nanobodies and antibodies. A great example for this are the dominant epitopes on SARS-CoV-2 RBDs.

    Growth curves (Fig. S3) show a large jump in recovery in growth under sybody induction conditions. Could the authors address this observation here and in the text?

    We suppose that this recovery represents suppressor mutants and/or (more likely) improved growth in the absence of functional Smc during nutrient limitation (see Gruber et al., 2013 and Wang et al., 2013). We will add this statement to the text.

    L41- Sentence correction: Loop can be removed. Ah, yes, sorry for this confusing error. Thank you.

    1. L525 - bsuSmc 'E' :extra E can be removed. To do. Thank you.
    2. References need to be properly formatted. To do. Thank you.
    3. The authors should add in figure legend for Fig 1i) details on representation of the purple region, and explain the grey strokes for orientation of the loop. To do.
    4. How many cells were analysed in the cell biological assays? Legends should include these information. To Be Included.

    Reviewer #1 (Significance (Required)):

    Overall, this is an impressive study that uses an elegant strategy to find inhibitors of protein activity in vivo. The manuscript is clearly written and the experiments are logical and well-designed. The findings from the study will be significant to the broad field of genome biology, synthetic biology and also SMC biology. Specifically, the coiled coil domain of SMC proteins have been proposed to be of high functional value. The authors have elegantly identified key coiled-coil regions that may be important for function, and parallelly exhibited potential of the use of synthetic sybody/designed binders for inhibition of protein activity.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    Review: "Single Domain Antibody Inhibitors Target the Coiled Coil Arms of the Bacillus subtilis SMC complex" by Ophélie Gosselin et al, Review Commons RC-2025-03280 Structural Maintenance of Chromosome proteins (SMCs), a family of proteins found in almost all organisms, are organizers of DNA. They accomplish this by a process known as loop extrusion, wherein double-stranded DNA is actively reeled in and extruded into loops. Although SMCs are known to have several DNA binding regions, the exact mechanism by which they facilitate loop extrusion is not understood but is believed to entail large conformational changes. There are currently several models for loop extrusion, including one wherein the coiled coil (CC) arms open, but there is a lack of insightful experimentation and analysis to confirm any of these models. The work presented aims to provide much-needed new tools to investigate these questions: conformation-selective sybodies (synthetic nanobodies) that are likely to alter the CC opening and closing reactions. The authors produced, isolated, and expressed sybodies that specifically bound to Bacillus subtilis Smc-ScpAB. Using chimeric Smc constructs, where the coiled coils were partly replaced with the corresponding sequences from Streptococcus pneumoniae, the authors revealed that the isolated sybodies all targeted the same 4N CC element of the Smc arms. This region is likely disrupted by the sybodies either by stopping the arms from opening (correctly) or forcing them to stay open (enough). Disrupting these functional elements is suggested to cause the Smc-dependent chromosome organization lethal phenotype, implying that arm opening and closing is a key regulatory feature of bacterial Smc-ScpAB. In summary, the authors present a new method for trapping bacterial Smc's in certain conformations using synthetic antibodies. Using these antibodies, they have pinpointed the (previously suggested) 4N region of the coiled coils as an essential site for the opening and closing of the Smc coiled coil arms and that hindering these reactions blocks Smc-driven chromosomal organization. The work has important implications for how we might elucidate the mechanism of DNA loop extrusion by SMC complexes. Some specific comments: Line 75: "likely stabilizing otherwise rare intermediates of the conformational cycle." - sorry, why is that being concluded? Why not stabilizing longer-lived oncformations? We will clarify this statement!

    Line 89: Sorry, possibly our lack of understanding: why first ribosome and then phage display?

    Ribosome display offers to screen around 10^12 sybodies per selection round (technically unrestricted library size), while for phage display, the library size is restricted to around 10^9 sybodies due to the fact that production of a phage library requires transformation of the phagemid plasmid into E. coli, thereby introducing a diversity bottleneck. This is why the sybody platform starts off with ribosome display. It switches to phage display from round 2 onwards because the output of the initial round of ribosome display is around 10^6 sybodies, which can be easily transferred into the phage display format. Phage display is used to minimize selection biases. For more information, please consult the original sybody paper (PMID: 29792401).

    Line 100: Why was only lethality selected? Less severe phenotypes not clear enough?

    Yes, colony size is more difficult to score robustly, as the sizes of individual transformant colonies can vary quite widely. The number of isolated sybodies was at the limit of further analysis.

    Line 106: Could it be tested somehow if convex and concave library sybodies fold in Bs?

    We did not focus on the non-functional sybody candidates and only sybodies of the loop library turned out to cause functional consequences at the cellular level. Notably, we will include gfp-imaging showing that non-lethal sybodies are expressed to similar levels that toxic sybodies. Given the identical scaffold of concave and loop sybodies (they only differ in their CDR3 length), we expect that the concave sybodies fold in the cytoplasm of B. subtilis. For the convex sybodies exhibiting a different scaffold, this will be tested.

    Line 125: Could Pxyl be repressed by glucose?

    To our knowledge and experience, repression by glucose (catabolite repression) does not work well in this context in B. subtilis.

    Line 131: The SMC replacement strain is a cool experiment and removes a lot of doubts!

    Thank you! (we agree 😊)

    Line 141: The mapping is good and looks reliable, but looks and feels like a tour de force? Of course, some cryo-EM would have been lovely (lines 228-229 understood, it has been tried!).

    Yes, we have made several attempts at structural biology. Unfortunately, Smc-ScpAB is not well suited for cryo-EM in our hands and crystallography with Smc fragments and sybodies did not yield well-diffracting crystals.

    Line 179: Mmmh. Do we not assume DNA binding on top of the dimerised heads to open the CC (clamp)?

    We will clarify the text here.

    Line 187: Having sybodies that presumably keep the CC together (closing) and some that do not allow them to come together correctly (opening) is really cool and probably important going forward.

    Thank you!

    Figure 1 Ai is not very colour-blind friendly.

    We are sorry for this oversight. We will try to make the color scheme more inclusive. Thank you for the notification.

    Optional: did the authors see any spontaneous mutations emerge that bypass the lethal phenotype of sybody expression?

    No, we did not observe spontaneous mutations suppressing the phenotype, possibly due to the limited number of cell generations observed. We tried to avoid suppressors by limiting growth, but this may indeed be a good future approach for further fine map the binding sites and to obtain insights into the mechanism of inhibition.

    Optional: we think it would be nice to try some biochemical experiment with BMOE/cysteine-crosslinked B. subtilis Smc in the mid-region (4N or next to it) of the Smc coiled coils to try to further strengthen the story. Some of the authors are experts in this technique and strains might already exist?

    We have indeed tried to study the impact of sybody binding on Smc conformation by cysteine cross-linking. However, we were not convinced by the results and thus prefer not to draw any conclusions from them. We will add a corresponding note to the text.

    Reviewer #2 (Significance (Required)):

    The authors present a new method for trapping bacterial Smc's in certain conformations using synthetic antibodies. Using these antibodies, they have pinpointed the (previously suggested) 4N region of the coiled coils as an essential site for the opening and closing of the Smc coiled coil arms and that hindering these reactions blocks Smc-driven chromosomal organization. The work has important implications for how we might elucidate the mechanism of DNA loop extrusion by SMC complexes. Thank you!

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    Gosselin et al. use the sybody technology to study effects of in vivo inhibition oft he Bacillus subtilis SMC complex. Smc proteins are central DNA binding elements of several complexes that are vital for chromosome dynamics in almost all organisms. Sybodies are selected from three different libraries of the single domain antibodies, using the „transition state" mutant Smc. They identify 14 such mutant sybodies that are lethal when expressed in vivo, because they prevent proper function of Smc. The authors present evidence suggesting that all obtained sybodies bind to a coiled-coil region close to the Smc „neck", and thereby interfere with the Smc activity cycle, as evidenced by defective ATPase activity when Smc is bound to DNA. The study is well done and presented and shows that the strategy is very potent in finding a means to quickly turn off a protein's function in vivo, much quicker than depleting the protein.

    The authors also draw conclusions on the molecular mode of action of the SMC complex. The provide a number of suggestive experiments, but in my view mostly indirect evidence for such mechanism.

    My main criticism ist hat the authors have used a single - and catalytically trapped form of SMC. They speculate why they only obtain sybodies from one library, and then only idenfity sybodies that bind to a rather small part oft he large Smc protein. While the approach is definitely valuable, it is biassed towards sybodies that bind to Smc in a quite special way, it seems. Using wild type Smc would be interesting, to make more robust statements about the action of sybodies potantially binding to different parts of Smc.

    As explained above, we are quite confident the Smc ATPase mutation did not bias the selection in an obvious way. The surprising bias towards coiled coil binding sites has likely other explanations, as they likely form a preferred epitope recognized by sybodies.

    Line 105: Alternatively, the other libraries did not produce good binders or these sybodies were 106 not stably expressed in B. subtilis. This could be tested using Western blotting - I am assuming sybody antibodies are commercially avalable. However, this test is not important for the overall study, it would just clarify a minor point.

    While there are antibody fragments available to augment the size of sybodies (PMID: 40108246), these recognize 3D-epitopes and are thus not suited for Western blotting. We did not follow up on the negative results much, but would like to point out again that there are several biases that likely emerge for the same reason (bias to library, bias to coiled coil binding site). If correct, then likely few other sybodies are effectively lethal in B. subtilis, with the exception of the ones isolated and characterized. We have added this notion to the manuscript. We have also tested the expression of non-lethal sybodies by gfp-tagging and imaging. These results will be included in the revision.

    Fig. 2B: is is odd to count Spo0J foci per cells, as it is clear from the images that several origins must be present within the fluorescent foci. I am fine with the „counting" method, as the images show there is a clear segregation defect when sybodies are expressed, I believe the authors should state, though, that this is not a replication block, but failure to segregate origins.

    We agree that this is an important point and will add a corresponding comment to the text.

    Testing binding sites of sybodies tot he SMC complex is done in an indirect manner, by using chimeric Smc constructs. I am surprised why the authors have not used in vitro crosslinking: the authors can purify Smc, and mass spectrometry analyses would identify sites where sybodies are crosslinked to Smc. Again, I am fine with the indirect method, but the authors make quite concrete statements on binding based on non-inhibition of chimeric Smc; I can see alternative explanations why a chimera may not be targeted.

    We have made several attempts of testing direct binding with mixed outcomes and decided to not include those results in the light of the stronger and more relevant in vivo mapping. However, we will add ELISA results and briefly discuss grating coupled interferometry (GCI) data and pull-downs.

    Smc-disrupting sybodies affect the ATPase activity in one of two ways. Again, rather indirect experiments. This leads to the point Revealing Smc arm dynamics through synthetic binders in the discussion. The authors are quite careful in stating that their experiments are suggestive for a certain mode of action of Smc, which is warranted.

    In line 245, they state More broadly, the study demonstrates how synthetic binders can trap, stabilize, or block transient conformations of active chromatin-associated machines, providing a powerful means to probe their mechanisms in living cells. This is off course a possible scenario for the use of sybodies, but the study does not really trap Smc in a transient conformation, at least this is not clearly shown.

    We agree and will carefully rephrase this statement. Thank you.

    Overall, it is an interesting study, with a well-presented novel technology, and a limited gain of knowledge on SMC proteins. We respectfully disagree with the last point, since our unique results highlight the importance of the Smc coiled coils, which are otherwise largely neglected in the SMC literature, likely (at least in part) due the mild effect of single point mutations on coiled coil dynamics.

    Reviewer #3 (Significance (Required)):

    The work describes the gaining and use of single-binder antibodies (sybodies) to interfere with the function of proteins in bacteria. Using this technology for the SMC complex, the authors demonstrate that they can obtain a significant of binders that target a defined region is SMC and thereby interfere with the ATPase cycle.

    The study does not present a strong gain of knowledge of the mode of action of the SMC complex.

    As pointed out above, we respectfully disagree with this assertion.

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.

    4. Description of analyses that authors prefer not to carry out

    Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

    As pointed out above, there are a few minor points that we prefer not to experimentally address. In particular, we do not consider it as necessary to determine the expression levels of sybodies which were non-inhibitory. We also wish to note that we attempted to obtain structural additional biochemical data and to that end performed cryo-EM, crystallography and cysteine cross-linking experiments. Unfortunately, we did not obtain sybody complex structures and the cross-linking data were unfortunately not conclusive. We also wish to note that the first author has finished her PhD and left the lab, which limits our capacity to add additional experiments. However, as the reviewers also pointed out, the main conclusions are well supported by the data already.

  9. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    Gosselin et al. use the sybody technology to study effects of in vivo inhibition oft he Bacillus subtilis SMC complex. Smc proteins are central DNA binding elements of several complexes that are vital for chromosome dynamics in almost all organisms. Sybodies are selected from three different libraries of the single domain antibodies, using the „transition state" mutant Smc. They identify 14 such mutant sybodies that are lethal when expressed in vivo, because they prevent proper function of Smc. The authors present evidence suggesting that all obtained sybodies bind to a coiled-coil region close to the Smc „neck", and thereby interfere with the Smc activity cycle, as evidenced by defective ATPase activity when Smc is bound to DNA. The study is well done and presented and shows that the strategy is very potent in finding a means to quickly turn off a protein's function in vivo, much quicker than depleting the protein.

    The authors also draw conclusions on the molecular mode of action of the SMC complex. The provide a number of suggestive experiments, but in my view mostly indirect evidence for such mechanism.

    My main criticism ist hat the authors have used a single - and catalytically trapped form of SMC. They speculate why they only obtain sybodies from one library, and then only idenfity sybodies that bind to a rather small part oft he large Smc protein. While the approach is definitely valuable, it is biassed towards sybodies that bind to Smc in a quite special way, it seems. Using wild type Smc would be interesting, to make more robust statements about the action of sybodies potantially binding to different parts of Smc.

    Line 105: Alternatively, the other libraries did not produce good binders or these sybodies were 106 not stably expressed in B. subtilis. This could be tested using Western blotting - I am assuming sybody antibodies are commercially avalable. However, this test is not important for the overall study, it would just clarify a minor point.

    Fig. 2B: is is odd to count Spo0J foci per cells, as it is clear from the images that several origins must be present within the fluorescent foci. I am fine with the „counting" method, as the images show there is a clear segregation defect when sybodies are expressed, I believe the authors should state, though, that this is not a replication block, but failure to segregate origins.

    Testing binding sites of sybodies tot he SMC complex is done in an indirect manner, by using chimeric Smc constructs. I am surprised why the authors have not used in vitro crosslinking: the authors can purify Smc, and mass spectrometry analyses would identify sites where sybodies are crosslinked to Smc. Again, I am fine with the indirect method, but the authors make quite concrete statements on binding based on non-inhibition of chimeric Smc; I can see alternative explanations why a chimera may not be targeted.

    Smc-disrupting sybodies affect the ATPase activity in one of two ways. Again, rather indirect experiments. This leads to the point Revealing Smc arm dynamics through synthetic binders in the discussion. The authors are quite careful in stating that their experiments are suggestive for a certain mode of action of Smc, which is warranted.

    In line 245, they state More broadly, the study demonstrates how synthetic binders can trap, stabilize, or block transient conformations of active chromatin-associated machines, providing a powerful means to probe their mechanisms in living cells. This is off course a possible scenario for the use of sybodies, but the study does not really trap Smc in a transient conformation, at least this is not clearly shown.

    Overall, it is an interesting study, with a well-presented novel technology, and a limited gain of knowledge on SMC proteins.

    Significance

    The work describes the gaining and use of single-binder antibodies (sybodies) to interfere with the function of proteins in bacteria. Using this technology for the SMC complex, the authors demonstrate that they can obtain a significant of binders that target a defined region is SMC and thereby interfere with the ATPase cycle.

    The study does not present a strong gain of knowledge of the mode of action of the SMC complex.

  10. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    Review: "Single Domain Antibody Inhibitors Target the Coiled Coil Arms of the Bacillus subtilis SMC complex" by Ophélie Gosselin et al, Review Commons RC-2025-03280

    Structural Maintenance of Chromosome proteins (SMCs), a family of proteins found in almost all organisms, are organizers of DNA. They accomplish this by a process known as loop extrusion, wherein double-stranded DNA is actively reeled in and extruded into loops. Although SMCs are known to have several DNA binding regions, the exact mechanism by which they facilitate loop extrusion is not understood but is believed to entail large conformational changes. There are currently several models for loop extrusion, including one wherein the coiled coil (CC) arms open, but there is a lack of insightful experimentation and analysis to confirm any of these models. The work presented aims to provide much-needed new tools to investigate these questions: conformation-selective sybodies (synthetic nanobodies) that are likely to alter the CC opening and closing reactions.

    The authors produced, isolated, and expressed sybodies that specifically bound to Bacillus subtilis Smc-ScpAB. Using chimeric Smc constructs, where the coiled coils were partly replaced with the corresponding sequences from Streptococcus pneumoniae, the authors revealed that the isolated sybodies all targeted the same 4N CC element of the Smc arms. This region is likely disrupted by the sybodies either by stopping the arms from opening (correctly) or forcing them to stay open (enough). Disrupting these functional elements is suggested to cause the Smc-dependent chromosome organization lethal phenotype, implying that arm opening and closing is a key regulatory feature of bacterial Smc-ScpAB. In summary, the authors present a new method for trapping bacterial Smc's in certain conformations using synthetic antibodies. Using these antibodies, they have pinpointed the (previously suggested) 4N region of the coiled coils as an essential site for the opening and closing of the Smc coiled coil arms and that hindering these reactions blocks Smc-driven chromosomal organization. The work has important implications for how we might elucidate the mechanism of DNA loop extrusion by SMC complexes.

    Some specific comments:

    Line 75: "likely stabilizing otherwise rare intermediates of the conformational cycle." - sorry, why is that being concluded? Why not stabilizing longer-lived oncformations?

    Line 89: Sorry, possibly our lack of understanding: why first ribosome and then phage display?

    Line 100: Why was only lethality selected? Less severe phenotypes not clear enough?

    Line 106: Could it be tested somehow if convex and concave library sybodies fold in Bs?

    Line 125: Could Pxyl be repressed by glucose?

    Line 131: The SMC replacement strain is a cool experiment and removes a lot of doubts!

    Line 141: The mapping is good and looks reliable, but looks and feels like a tour de force? Of course, some cryo-EM would have been lovely (lines 228-229 understood, it has been tried!).

    Line 179: Mmmh. Do we not assume DNA binding on top of the dimerised heads to open the CC (clamp)?

    Line 187: Having sybodies that presumably keep the CC together (closing) and some that do not allow them to come together correctly (opening) is really cool and probably important going forward.

    Figure 1 Ai is not very colour-blind friendly. Optional: did the authors see any spontaneous mutations emerge that bypass the lethal phenotype of sybody expression?

    Optional: we think it would be nice to try some biochemical experiment with BMOE/cysteine-crosslinked B. subtilis Smc in the mid-region (4N or next to it) of the Smc coiled coils to try to further strengthen the story. Some of the authors are experts in this technique and strains might already exist?

    Significance

    The authors present a new method for trapping bacterial Smc's in certain conformations using synthetic antibodies. Using these antibodies, they have pinpointed the (previously suggested) 4N region of the coiled coils as an essential site for the opening and closing of the Smc coiled coil arms and that hindering these reactions blocks Smc-driven chromosomal organization. The work has important implications for how we might elucidate the mechanism of DNA loop extrusion by SMC complexes.

  11. Review Commons

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    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Gosselin et al., develop a method to target protein activity using synthetic single-domain nanobodies (sybodies). They screen a library of sybodies using ribosome/ phage display generated against bacillus Smc-ScpAB complex. Specifically, they use an ATP hydrolysis deficient mutant of SMC so as to identify sybodies that will potentially disrupt Smc-ScpAB activity. They next screen their library in vivo, using growth defects in rich media as a read-out for Smc activity perturbation. They identify 14 sybodies that mirror smc deletion phenotype including defective growth in fast-growth conditions, as well as chromosome segregation defects. The authors use a clever approach by making chimeras between bacillus and S. pnuemoniae Smc to narrow-down to specific regions within the bacillus Smc coiled-coil that are likely targets of the sybodies. Using ATPase assays, they find that the sybodies either impede DNA-stimulated ATP hydrolysis or hyperactivate ATP hydrolysis (even in the absence of DNA). The authors propose that the sybodies may likely be locking Smc-ScpAB in the "closed" or "open" state via interaction with the specific coiled-coil region on Smc. I have a few comments that the authors should consider:

    Major comments:

    1. Lack of direct in vitro binding measurements: The authors do not provide measurements of sybody affinities, binding/ unbinding kinetics, stoichiometries with respect to Smc-ScpAB. Additionally, do the sybodies preferentially interact with Smc in ATP/ DNA-bound state? And, do the sybodies affect the interaction of ScpAB with SMC? It is understandable that such measurements for 14 sybodies is challenging, and not essential for this study. Nonetheless, it is informative to have biochemical characterization of sybody interaction with the Smc-ScpAB complex for at least 1-2 candidate sybodies described here.
    2. Many modes of sybody binding to Smc are plausible The authors provide an elaborate discussion of sybodies locking the Smc-ScpAB complex in open/ closed states. However, in the absence of structural support, the mechanistic inferences may need to be tempered. For example, is it also not possible for the sybodies to bind the inner interface of the coiled-coil, resulting in steric hinderance to coiled-coil interactions. It is also possible that sybody interaction disrupts ScpAB interaction (as data ruling this possibility out has not been provided). Thus, other potential mechanisms would be worth considering/ discussing. In this direction, did AlphaFold reveal any potential insights into putative binding locations?
    3. Sybody expression in vivo Have the authors estimated sybody expression in vivo? Are they all expressed to similar levels?
    4. Sybodies should phenocopy ATP hydrolysis mutant of Smc The sybodies were screened against an ATP hydrolysis deficient mutant of Smc, with the rationale that these sybodies would interfere this step of the Smc duty cycle. Does the expression of the sybodies in vivo phenocopy the ATP hydrolysis deficient mutant of Smc? Could the authors consider any phenotypic read-outs that can indicate whether the sybody action results in an smc-null effect or specifically an ATP hydrolysis deficient effect?

    Minor comments:

    1. It was surprising that no sybodies were found that could target both bacillus and spneu Smc. For example, sybodies targeting the head regions of Smc that might work in a more universal manner. Could the authors comment on the coverage of the sybodies across the protein structure?
    2. Growth curves (Fig. S3) show a large jump in recovery in growth under sybody induction conditions. Could the authors address this observation here and in the text?
    3. L41- Sentence correction: Loop can be removed.
    4. L525 - bsuSmc 'E' :extra E can be removed.
    5. References need to be properly formatted.
    6. The authors should add in figure legend for Fig 1i) details on representation of the purple region, and explain the grey strokes for orientation of the loop.
    7. How many cells were analysed in the cell biological assays? Legends should include these information.

    Significance

    Overall, this is an impressive study that uses an elegant strategy to find inhibitors of protein activity in vivo. The manuscript is clearly written and the experiments are logical and well-designed. The findings from the study will be significant to the broad field of genome biology, synthetic biology and also SMC biology. Specifically, the coiled coil domain of SMC proteins have been proposed to be of high functional value. The authors have elegantly identified key coiled-coil regions that may be important for function, and parallelly exhibited potential of the use of synthetic sybody/designed binders for inhibition of protein activity.

  12. Version published to 10.1101/2025.10.16.682983 on bioRxiv