Competitive binding of MatP and topoisomerase IV to the MukB dimerization hinge

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1. 

   eLife

   Sep 20, 2021

   Author Response:

   Reviewer #1 (Public Review):

   The authors bring compelling biochemical evidence that MatP and ParC compete to interact with the hinge of MukB. In addition, the authors made an effort to support their hypothesis with the description of the phenotype of a mutant of the hinge, mukBKKK, which fails to interact with both ParC and MatP.

   Thank you for your positive response!

   Reviewer #2 (Public Review):

   In the manuscript entitled "Competitive binding of MatP and Topoisomerase IV to the MukB hinge modulates chromosome organization-segregation", Fisher and colleagues characterized in vitro the interaction between MukB and MatP or between MukB and TopoIV using different biochemical approaches. First, they identified that a dimer of MatP or a dimer ParC interacts in vitro with the hinge domain of MukB but failed to form a …

   Author Response:

   Reviewer #1 (Public Review):

   The authors bring compelling biochemical evidence that MatP and ParC compete to interact with the hinge of MukB. In addition, the authors made an effort to support their hypothesis with the description of the phenotype of a mutant of the hinge, mukBKKK, which fails to interact with both ParC and MatP.

   Thank you for your positive response!

   Reviewer #2 (Public Review):

   In the manuscript entitled "Competitive binding of MatP and Topoisomerase IV to the MukB hinge modulates chromosome organization-segregation", Fisher and colleagues characterized in vitro the interaction between MukB and MatP or between MukB and TopoIV using different biochemical approaches. First, they identified that a dimer of MatP or a dimer ParC interacts in vitro with the hinge domain of MukB but failed to form a tripartite complex ParC/MatP/MukB. Second, they observed that the interaction of full-length protein MatP with MukB hinge is competed out by ParC suggesting that MatP and ParC share overlapping binding sites on the MukB hinge. Third, using a MukB mutant (MukB-kkk) known to be deficient in ParC binding, the authors did not detect any major topo-IV defective phenotype questioning the requirement of direct interaction between TopoIV and MukBEF for chromosome unlinking by decatenation.

   Several major difficulties regarding this manuscript are indicated below:

   1. Although the authors have thoroughly and elegantly analyzed the in vitro interactions of the MukB hinge with either MatP or ParCCTD, there is no strong evidence revealing the significance of these interactions in vivo. While the interaction between MukB hinge and ParC has been reported by several groups over the years using two-hybrid screens and pull-down assays, the data presented in this manuscript reveal that the absence of such interactions in vivo do not cause major defects in decatenation/chromosome segregation resulting from an impaired action of TopoIV and do not importantly affect MukBEF activity.

   The above statement is largely correct, but in our opinion does not detract from the ‘elegant’ and ‘thorough’ work in the manuscript! We note that as here, most science advances are incremental. Using the methodologies available, we have not uncovered the detailed mechanism or significance of the hinge-MatP interaction in vivo and how it relates to the recently discovered second binding site for MatP-matS in the MukB coiled-coils. The relationship between the different MatP-bound states revealed by Bürmann and colleagues and ourselves sets the scene for future work by others! Furthermore, the new analyses clearly show that MukBKKK expressing cells have a phenotype more closely related to that of ΔmukB cells than ΔmatP cells, consistent with the second MatP binding site (in the presence of matS DNA in the hydrolysis-impaired ‘locked’ state of MukBEQ). In relation to the significance of the TopoIV/ParC-MukB hinge interaction, it is already clear from the literature that in the complete absence of MukBEF, there is sufficient topoIV activity in cells to allow relatively normal growth chromosome segregation during growth in minimal medium or in rich medium at low temperature. At restrictive temperature ΔmukB cells are filamentous and have unsegregated nucleoids. Prior to this work, we had proposed that much of the ΔmukB phenotype, including temperature-sensitive growth in rich medium as well as delayed decatenation of new replicated oris was related to the knock-on effects of mis-localising and mismanaging TopoIV recruitment to chromosomes, and in particular ori. Our analyses show that MukBKKKEF expressing cells may have a modest defect in decatenation at ori, as measured by an increased fraction of cells with unsegregated oris; intermediate between WT and ΔmukB cells, but this does not lead to temperature-sensitivity (Figure 5 and its Supplementary Figure).

   1. A robust readout is required to demonstrate the significance of the MatP-MukBEF interaction in vivo. The read-out used in this study (morphology of MukBEF foci) to reveal the absence of interaction between MatP and MukBEF is not easily quantifiable. Different methods have been used before that revealed the inhibition/displacement of MukBEF by MatP. Such methods (Mäkela and Sherratt, 2020; Lioy et al., 2018; 2020) should be used to estimate the MukBEF positioning/activity in the absence of interaction with MatP.

   In the revised manuscript, we have since extended our analyses to explore further the properties of the MukBKKK mutant and the significance of the MatP-MukBEF interaction in vivo, and state what conclusions arise from the new analyses. Elsewhere in this response, we explain why Hi-C, ChIP-Seq and SIM would not be helpful in our opinion. We did not have the reagents to undertake PALM by the time our group disbanded. In our opinion, the information from PALM would be helpful in ascertaining more about the behavior of MukBKKKEF in cells, but that is beyond the scope of this work.

   1. The in vivo interaction of MatP with the MukB hinge revealed by the bacterial two-hybrid assay (Nolivos et al., 2016) seems to be weak. A recent study (Bürmann et al. BioRxiv) has revealed the CryoEM structure of MukBEF complexed with MatP. In the structure obtained, MatP bound to its target matS interacts with MukE and the "joint"region of MukB, not with the hinge. Based on the structure, Bürmann and colleagues propose a very attractive unloading model enhanced at matS sites, explaining how MatP prevents MukBEF activity in the Ter region. In the present manuscript, it is not addressed whether the interaction of MatP with the hinge corresponds to a subsequent stage during unloading or contributes to another aspect of MukBEF activity.

   We agree with the above. At the time of completing our experimental work we became aware of the work of Bürmann and colleagues, but it was not in the public domain and therefore not suitable for discussion in the original manuscript. Since that work has now been deposited on bioRxiv, we now discuss it and how it relates to our own work. There is nothing inconsistent between the two complementary sets of work. Together they support the idea, proposed in the original manuscript, that the MatP binding to the hinge that we characterize is just one stage in a multi-step reaction, and provide the platform for future studies.

   1. DNA entrapment by MukBEF requires ATP hydrolysis (Bürmann et al. BioRxiv). It is therefore not obvious to understand how MukBEFEQ is bound in vivo to matS sites. Also as matS sites compete with the MukB hinge for MatP binding in vitro, it is not clear how MukBEF could interact with MatP bound to matS sites in vivo.

   EQ mutants of multiple SMC complexes studied load onto chromosomes in vivo, (imaging; ChIP-Seq etc.), where they remain stably associated, turning over slowly. In-line with this, MukBEQ mutants stably associate with chromosomal DNA (and accumulate at MatP-matS sites), where they have very slow off rates (e.g. Science, 338, 528–31). The DNA entrapment assay of Burmann will trap some types of stably bound MukBEF complexes (‘topologically entrapped’), but not others, as has been shown for several different SMC complexes. Importantly, ATP hydrolysis is required to release EQ SMC complexes from chromosomes (whether it be MukBEF or others), assayed in many different systems and published in many places. With respect to the second sentence of the reviewer, the second binding site for MatP-matS provides a means for how MukBEF interacts with MatP-matS in vivo. The revised manuscript clarifies all of this.

   1. It is not clear how matS sites could prevent MatP-MukB in vitro interaction. This is not consistent with the observation that MatP is required bound at matS sites to unload/prevent MukBEF activity.

   This is fully discussed in the revised manuscript.

   1. The title is misleading as no clear evidence is reported concerning an effect of the interactions on chromosome organization/segregation.

   We have modified the title to take account of this point: ‘Competitive binding of MatP and topoisomerase IV to the MukB hinge domain’.

   Reviewer #3 (Public Review):

   This paper focuses on protein-protein interactions of the E.coli Structural Maintenance of Chromosomes (SMC) complex MukBEF through the hinge domain of its MukB subunit. Previous work has demonstrated that the MukB hinge interacts with the ParC subunit of topoisomerase IV (ParC2E2), which decatenates sister chromosomes, and the MatP homodimer, which preferentially binds chromosomal matS sites near the replication terminus (ter) and excludes MukBEF from ter. This paper expands on previous studies by using a wide range of in vitro assays (isothermal titration calorimetry, native mass spectrometric, fluorescence correlation spectroscopy, analytical size exclusion chromatography, etc) to characterize MukB-MatP and MukB-ParC interactions qualitatively and quantitatively. One major finding is that MatP and ParC compete for MukB hinge binding, rather than forming a MukB-MatP-ParC ternary complex. Additionally, this study reports that a ParC-binding deficient MukB mutant (MukBKKK) is also deficient in MatP binding, suggesting that ParC and MatP have overlapping binding sites on MukB. Further, MatP-matS binding prevents MatP from binding MukB, suggesting that MukB and matS have overlapping binding sites on MatP. Live-cell fluorescence imaging of WT MukB and the binding-deficient MukBKKK mutant confirm that MukBKKK does not colocalize preferentially with ori or bind ParC, in contrast to WT MukB, although it does not show some of the expected Muk˗ phenotypes such as temperature-sensitive growth.

   Strengths of this study:

   1. Using a range of experimental techniques to study binding interactions between MukB, ParC/topo IV, and MatP helps increase confidence in the findings. For example, multiple lines of evidence (analytical size exclusion chromatography, native mass spectrometry, and fluorescence correlation spectroscopy) all indicate that there is no or minimal formation of a MukB-MatB-ParC ternary complex and that instead MatP and ParC bind competitively to MukB.

   1. The in vitro assays use only partially reconstituted complexes, including a substantially truncated form of MukB containing the hinge and a portion of the coiled-coil domain. The inclusion of in vivo imaging experiments showing that the binding-deficient MukBKKK mutant is impaired in ParC binding and proper localization at ori helps to support the relevance of the in vitro results.

   1. Experiments are well-controlled and the use of a number of MukB, ParC, and MatP mutants helps to support the conclusions regarding the interactions between these proteins.

   Thank you for the above.

   Weaknesses of this study:

   1. In some cases, different experimental techniques are used to measure binding interactions between WT and mutant proteins, and no explanation is given for the choice of technique. For example, isothermal titration calorimetry and native mass spectrometry are initially used to measure the MukB hinge binding to MatP, but these techniques are not used to characterize the binding of the MukBKKK hinge mutant to MatP. Comparisons between WT MukB and MukBKKK binding to MatP are provided by other methods (native PAGE, fluorescence correlation spectroscopy, analytical size exclusion chromatography), so the difference in binding between the WT and mutant is clearly demonstrated, but the lack of these corresponding experiments creates some confusion and makes it more challenging to interpret some of the results.

   We have clarified the text to ease the reader through the assortment of biochemical and biophysical techniques utilised. Our choice of techniques in part reflected the different personnel and equipment available over the several years that this project spanned and also the properties and sizes of the complexes under study; these influence the analytical technique chosen. We believe that the quantitative data re. stoichiometries and KDs etc. are directly comparable between the different analyses. We would argue that the range of techniques and comparisons between analytical tools is a strength rather than a weakness.

   1. Although the experiments and analyses are for the most part rigorous and well-controlled, there are a few minor experiments or analyses with some weaknesses. In the analysis of cell size in the in vivo imaging experiments (Figure 5D), the median cell lengths are reported for WT MukB and different mutants. The authors conclude that the cell size is essentially the same for WT MukB and the binding-deficient MukBKKK mutant, suggesting no major chromosome segregation defects for the mutant. However, the difference in median cell length between WT and MukBKKK is the same as that between WT and ΔmukB cells. Thus it is not clear how the authors draw their conclusion from these data. Further discussion and analysis (perhaps the presentation of the full distribution of cell lengths and/or statistical analysis) might support these claims. Further, the demonstration that the MukBS781F mutant is not growth-sensitive (Supplementary Figure 3C) appears to represent a single experiment, whereas even a second replicate would help increase confidence in the results.

   We now present the distributions of median cell lengths (Supplementary Figure 5A). It is true that there is no significant difference in cell length distributions of WT, mukB and mukBKKK cells grown at 30oC in minimal medium, leading to the conclusion that none of these strains have a major cell division defect under these conditions. This is all clarified in the revised text.

   The complementation assay presented in Supplementary Figure 3C is indeed from a single repeat. Use of a Phe substitution at position S718 was intended to give an indication as to whether substitution of residue S718 with 4-azido-L-phenylalanine for unnatural amino acid (UAA) labelling impeded MukB activity (something not tractable to unequivocally test for in vivo). Following purification of the UAA-containing protein, subsequent labelling and analysis of its ATP hydrolysis activity and its in vitro binding to MukEF, ParC and MatP, it was clear this protein retained its assayable functions. We are happy to remove this in vivo complementation if the reviewer wishes - it does not impact the quality of the experiments with the fluorophore-labelled protein.

   1. Although this study provides a more in-depth characterization of the ParC/topo IV and MatP interactions with the MukB hinge than in previous work, it is not clear that the results lead to substantial changes in our understanding of the role of these protein-protein interactions in coordinating chromosome segregation.

   Prior to this work, the binding interface of MatP upon MukB had not been mapped other than at the domain level (Nolivos et al. 2016). The work presented here builds upon this and pinpoints a refined patch upon the MukB hinge domain essential for MatP interaction. Moreover, this same surface is bound by ParC indicating likely competition for binding, something previously only alluded to by in vivo studies but not yet demonstrated in vitro. Both of these revelations, alongside the important demonstration that a single topoIV heterotetramer binds a dimeric hinge, reinforce a model in which MukBEF activity is regulated in a spatial and temporal manner by binding of its partner proteins, MatP and TopoIV, which is reliant upon the competitive nature of their binding to MukB.

   Read the original source
2. 

   eLife

   Sep 20, 2021

   Evaluation Summary:

   This paper is of potential interest to an audience of biochemists, cell biologists, and structural biologists working in the area of chromosome organization and segregation. A wide range of in vitro methods is used to provide compelling biochemical evidence for the interaction of MatP and ParEC at the hinge of MukB, the condensin of Enterobacteria. However, the evidence supporting the significance of these interactions in vivo is less strong, limiting the biological implications of the elegant biochemical findings.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

   Read the original source
3. 

   eLife

   Sep 20, 2021

   Reviewer #1 (Public Review):

   The authors bring compelling biochemical evidence that MatP and ParC compete to interact with the hinge of MukB. In addition, the authors made an effort to support their hypothesis with the description of the phenotype of a mutant of the hinge, mukBKKK, which fails to interact with both ParC and MatP.

   Read the original source
4. 

   eLife

   Sep 20, 2021

   Reviewer #2 (Public Review):

   In the manuscript entitled "Competitive binding of MatP and Topoisomerase IV to the MukB hinge modulates chromosome organization-segregation", Fisher and colleagues characterized in vitro the interaction between MukB and MatP or between MukB and TopoIV using different biochemical approaches. First, they identified that a dimer of MatP or a dimer ParC interacts in vitro with the hinge domain of MukB but failed to form a tripartite complex ParC/MatP/MukB. Second, they observed that the interaction of full-length protein MatP with MukB hinge is competed out by ParC suggesting that MatP and ParC share overlapping binding sites on the MukB hinge. Third, using a MukB mutant (MukB-kkk) known to be deficient in ParC binding, the authors did not detect any major topo-IV defective phenotype questioning the requirement …

   Reviewer #2 (Public Review):

   In the manuscript entitled "Competitive binding of MatP and Topoisomerase IV to the MukB hinge modulates chromosome organization-segregation", Fisher and colleagues characterized in vitro the interaction between MukB and MatP or between MukB and TopoIV using different biochemical approaches. First, they identified that a dimer of MatP or a dimer ParC interacts in vitro with the hinge domain of MukB but failed to form a tripartite complex ParC/MatP/MukB. Second, they observed that the interaction of full-length protein MatP with MukB hinge is competed out by ParC suggesting that MatP and ParC share overlapping binding sites on the MukB hinge. Third, using a MukB mutant (MukB-kkk) known to be deficient in ParC binding, the authors did not detect any major topo-IV defective phenotype questioning the requirement of direct interaction between TopoIV and MukBEF for chromosome unlinking by decatenation.

   Several major difficulties regarding this manuscript are indicated below:

   1. Although the authors have thoroughly and elegantly analyzed the in vitro interactions of the MukB hinge with either MatP or ParCCTD, there is no strong evidence revealing the significance of these interactions in vivo. While the interaction between MukB hinge and ParC has been reported by several groups over the years using two-hybrid screens and pull-down assays, the data presented in this manuscript reveal that the absence of such interactions in vivo do not cause major defects in decatenation/chromosome segregation resulting from an impaired action of TopoIV and do not importantly affect MukBEF activity.

   1. A robust readout is required to demonstrate the significance of the MatP-MukBEF interaction in vivo. The read-out used in this study (morphology of MukBEF foci) to reveal the absence of interaction between MatP and MukBEF is not easily quantifiable. Different methods have been used before that revealed the inhibition/displacement of MukBEF by MatP. Such methods (Mäkela and Sherratt, 2020; Lioy et al., 2018; 2020) should be used to estimate the MukBEF positioning/activity in the absence of interaction with MatP.

   1. The in vivo interaction of MatP with the MukB hinge revealed by the bacterial two-hybrid assay (Nolivos et al., 2016) seems to be weak. A recent study (Bürmann et al. BioRxiv) has revealed the CryoEM structure of MukBEF complexed with MatP. In the structure obtained, MatP bound to its target matS interacts with MukE and the "joint"region of MukB, not with the hinge. Based on the structure, Bürmann and colleagues propose a very attractive unloading model enhanced at matS sites, explaining how MatP prevents MukBEF activity in the Ter region. In the present manuscript, it is not addressed whether the interaction of MatP with the hinge corresponds to a subsequent stage during unloading or contributes to another aspect of MukBEF activity.

   1. DNA entrapment by MukBEF requires ATP hydrolysis (Bürmann et al. BioRxiv). It is therefore not obvious to understand how MukBEFEQ is bound in vivo to matS sites. Also as matS sites compete with the MukB hinge for MatP binding in vitro, it is not clear how MukBEF could interact with MatP bound to matS sites in vivo.

   1. It is not clear how matS sites could prevent MatP-MukB in vitro interaction. This is not consistent with the observation that MatP is required bound at matS sites to unload/prevent MukBEF activity.

   1. The title is misleading as no clear evidence is reported concerning an effect of the interactions on chromosome organization/segregation.

   Read the original source
5. 

   eLife

   Sep 20, 2021

   Reviewer #3 (Public Review):

   This paper focuses on protein-protein interactions of the E.coli Structural Maintenance of Chromosomes (SMC) complex MukBEF through the hinge domain of its MukB subunit. Previous work has demonstrated that the MukB hinge interacts with the ParC subunit of topoisomerase IV (ParC2E2), which decatenates sister chromosomes, and the MatP homodimer, which preferentially binds chromosomal matS sites near the replication terminus (ter) and excludes MukBEF from ter. This paper expands on previous studies by using a wide range of in vitro assays (isothermal titration calorimetry, native mass spectrometric, fluorescence correlation spectroscopy, analytical size exclusion chromatography, etc) to characterize MukB-MatP and MukB-ParC interactions qualitatively and quantitatively. One major finding is that MatP and ParC …

   Reviewer #3 (Public Review):

   This paper focuses on protein-protein interactions of the E.coli Structural Maintenance of Chromosomes (SMC) complex MukBEF through the hinge domain of its MukB subunit. Previous work has demonstrated that the MukB hinge interacts with the ParC subunit of topoisomerase IV (ParC2E2), which decatenates sister chromosomes, and the MatP homodimer, which preferentially binds chromosomal matS sites near the replication terminus (ter) and excludes MukBEF from ter. This paper expands on previous studies by using a wide range of in vitro assays (isothermal titration calorimetry, native mass spectrometric, fluorescence correlation spectroscopy, analytical size exclusion chromatography, etc) to characterize MukB-MatP and MukB-ParC interactions qualitatively and quantitatively. One major finding is that MatP and ParC compete for MukB hinge binding, rather than forming a MukB-MatP-ParC ternary complex. Additionally, this study reports that a ParC-binding deficient MukB mutant (MukBKKK) is also deficient in MatP binding, suggesting that ParC and MatP have overlapping binding sites on MukB. Further, MatP-matS binding prevents MatP from binding MukB, suggesting that MukB and matS have overlapping binding sites on MatP. Live-cell fluorescence imaging of WT MukB and the binding-deficient MukBKKK mutant confirm that MukBKKK does not colocalize preferentially with ori or bind ParC, in contrast to WT MukB, although it does not show some of the expected Muk˗ phenotypes such as temperature-sensitive growth.

   Strengths of this study:

   1. Using a range of experimental techniques to study binding interactions between MukB, ParC/topo IV, and MatP helps increase confidence in the findings. For example, multiple lines of evidence (analytical size exclusion chromatography, native mass spectrometry, and fluorescence correlation spectroscopy) all indicate that there is no or minimal formation of a MukB-MatB-ParC ternary complex and that instead MatP and ParC bind competitively to MukB.

   2. The in vitro assays use only partially reconstituted complexes, including a substantially truncated form of MukB containing the hinge and a portion of the coiled-coil domain. The inclusion of in vivo imaging experiments showing that the binding-deficient MukBKKK mutant is impaired in ParC binding and proper localization at ori helps to support the relevance of the in vitro results.

   3. Experiments are well-controlled and the use of a number of MukB, ParC, and MatP mutants helps to support the conclusions regarding the interactions between these proteins.

   Weaknesses of this study:

   1. In some cases, different experimental techniques are used to measure binding interactions between WT and mutant proteins, and no explanation is given for the choice of technique. For example, isothermal titration calorimetry and native mass spectrometry are initially used to measure the MukB hinge binding to MatP, but these techniques are not used to characterize the binding of the MukBKKK hinge mutant to MatP. Comparisons between WT MukB and MukBKKK binding to MatP are provided by other methods (native PAGE, fluorescence correlation spectroscopy, analytical size exclusion chromatography), so the difference in binding between the WT and mutant is clearly demonstrated, but the lack of these corresponding experiments creates some confusion and makes it more challenging to interpret some of the results.

   2. Although the experiments and analyses are for the most part rigorous and well-controlled, there are a few minor experiments or analyses with some weaknesses. In the analysis of cell size in the in vivo imaging experiments (Figure 5D), the median cell lengths are reported for WT MukB and different mutants. The authors conclude that the cell size is essentially the same for WT MukB and the binding-deficient MukBKKK mutant, suggesting no major chromosome segregation defects for the mutant. However, the difference in median cell length between WT and MukBKKK is the same as that between WT and ΔmukB cells. Thus it is not clear how the authors draw their conclusion from these data. Further discussion and analysis (perhaps the presentation of the full distribution of cell lengths and/or statistical analysis) might support these claims. Further, the demonstration that the MukBS781F mutant is not growth-sensitive (Supplementary Figure 3C) appears to represent a single experiment, whereas even a second replicate would help increase confidence in the results.

   3. Although this study provides a more in-depth characterization of the ParC/topo IV and MatP interactions with the MukB hinge than in previous work, it is not clear that the results lead to substantial changes in our understanding of the role of these protein-protein interactions in coordinating chromosome segregation.

   Read the original source
6. 

   Version published to 10.1101/2021.03.03.433707v1 on bioRxiv

   Mar 3, 2021