Mechanistic insights into GTP-dependence and kinetic polarity of FtsZ filament assembly

  1. Joyeeta Chakraborty
  2. Sakshi Poddar
  3. Soumyajit Dutta
  4. Vaishnavi Bahulekar
  5. Shrikant Harne
  6. Ramanujam Srinivasan
  7. Pananghat Gayathri

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

FtsZ, the tubulin homolog essential for bacterial cell division, assembles as Z-ring at the division site, and directs peptidoglycan synthesis by treadmilling. A key unanswered question is how FtsZ achieves its kinetic polarity that drives treadmilling. To obtain insights into fundamental features of FtsZ assembly dynamics independent of peptidoglycan synthesis, we report the characterization of FtsZ from the cell wall-less bacteria, Spiroplasma melliferum (SmFtsZ). SmFtsZ is a slower GTPase and has higher critical concentration (CC) for polymerization compared to Escherichia coli FtsZ (EcFtsZ). Analysis of the crystal structures of FtsZ structures reveal that the interaction of gamma phosphate of the nucleotide with the T3 loop leads to a peptide flip at Gly71. We propose that the flipped peptide conformation results in a key interaction that facilitates preferential binding of the N-terminal domain (NTD) of a GTP-bound FtsZ monomer to the C-terminal domain (CTD) exposed end of FtsZ filament. In FtsZs, a conformational switch from R- to T-state favors polymerization. We identified a residue, Phe224, located at the interdomain cleft of SmFtsZ, which is crucial for R- to T-state transition. The mutation F224M in SmFtsZ cleft resulted in higher GTPase activity and lower CC, whereas the corresponding M225F in EcFtsZ resulted in cell division defects in E. coli . Our results demonstrate that relative rotation of the domains is a rate-limiting step of polymerization. This step, in addition to the GTP-dependence of the T3 loop conformation, slows down the addition of monomers to the NTD-exposed end of filament in comparison to CTD end, thus explaining kinetic polarity.

Article activity feed

  1. Version published to 10.1101/2022.10.13.512043v4 on bioRxiv
  2. Version published to 10.1101/2022.10.13.512043v3 on bioRxiv
  3. Review Commons

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

    Learn more at Review Commons

    Reply to the reviewers

    The authors do not wish to provide a response at this time.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    Chakraborty et al describe the biochemical and structural characterization of Spiroplasma FtsZ and report that the protein has unusual properties compared to other FtsZ. Sedimentation and GTPase measurements showed that whereas the wild-type protein has a high critical concentration and low GTPase activity, a mutant predicted to facilitate FtsZ cleft opening (F224M) exhibited lower critical concentration and higher GTPase activity. In addition, the crystal structures of both wild-type and F224M SmFtsZ revealed a unique domain-swapped dimer configuration in which one of the monomers in each dimer exhibited an R/T hybrid or intermediate conformation, with the NTD in the T state and the CTD in the R state. The T state of FtsZ has only been observed before when the protein crystallizes as filaments. Thus, the crystal structure of SmFtsZ - which is not assembled in filaments - was interpreted as capturing a conformational state that could explain the kinetic polarity of FtsZ (preferential addition of subunits to the CTD-exposed end of FtsZ filament).

    This is a good quality manuscript overall, but which could still be improved by the suggestions below. In terms of significance, it provides new data to support current models for FtsZ assembly mechanism but no major new insights. The findings are interesting for a more specialized audience.

    Major points

    1. The peculiar biochemical properties of SmFtsZ (high CC, low GTPase) are well documented and interesting but deserve further critical assessment to rule out artifacts. The EM in Fig 1B suggests abundant aggregated protein (not monomers), in addition to filament bundles, which suggests that SmFtsZ is not stable under the experimental conditions used. There are reports that some FtsZ will lose nucleotide during purification and become partially unfolded and unstable (doi.org/10.1111/febs.15235). Figure S1E suggests that the same may be happening here, as the amount of GDP released by SmFtsZ seems to be lower than expected if all the protein had nucleotide. Perhaps the authors should repeat their experiments with SmFtsZ purified in the presence of GDP, which should stabilize the protein, to confirm that the biochemical properties of the protein stay the same.
    2. Another unexpected observation is that the SmFtsZ bundles are quite short despite the low GTPase activity of the protein, whereas mutant F224M forms much longer bundles and is a stronger GTPase. In general, filament length correlates inversely with GTPase activity, if measurements are being made at steady state. However, no kinetic (light scattering or fluorescence) experiments seem to have been done to ensure measurements were done in steady state. The authors do try to explain the odd behavior of SmFtsZ but the idea that the increase in GTPase reflects a faster kinetics of nucleation and elongation is not necessarily true. GTP turnover is usually limited by the kinetics of filament disassembly not by assembly. However, it is possible that in reactions with a mutant that is much better at nucleation there will be many more filaments than with a poorly nucleating protein and, thus, more filament ends for subunit turnover. A complicator to these experiments is that they were carried out in high magnesium and at pH 6.5 which favor bundling, and bundling affects subunit and GTP turnover in ways that are hard to account for. Ideally, experiments aimed at properly determining the kinetic properties of FtsZ should be carried out under conditions that avoid bundling (pH 7.4-7.7, 2-5 mM Mg2+) and include proper kinetic measurements, such as light scattering. Thus, before any hard conclusions can be drawn about the properties of SmFtsZ, the authors may wish to revisit some of their biochemical experiments in light of the caveats pointed out here.
    3. A central part of the paper is the description of the intermediate R/T conformation but that was a bit confusing and perhaps could be improved. The first thing would be to more clearly define what are the structural changes of the NTD in the T conformation. From other publications, it seems that the NTD undergoes little alteration upon switching to the T conformation, the main one being the flipping of the guanine of the bound nucleotide. But if the NTD structure remains essentially the same, what causes the flipping of the guanine? My impression was that guanine flipping was caused by the downward movement of H7 but if H7 and its attached elements (H6, S6) are moving, why is this not manifested as a significant structural change in the NTD in the T state? Moreover, from Figs. 3C and 5A we conclude that the relative position of H7 in the R/T structures is the same as in R structures. If H7 has not changed in the R/T structure, can you call this a T structure? Also, if there is no H7 movement, what caused the change in guanine angle?
    4. The observation that the intermediate conformation was detected in a swapped-dimer is always a matter of some concern, as domain swapping imposes additional constraints on the conformational freedom of a protein and generates structures that are often different from their non-swapped counterparts. This seems to be the case for other FtsZ domain-swapped structures, which were outliers in the extensive comparisons made by Wagstaff et al (doi.org/10.1128/mBio.00254-17) and also stand out in the analysis in Fig. 3BC. Perhaps the authors should discuss more thoroughly why this structure must reflect a natural conformation of FtsZ.
    5. Still regarding the structural basis of kinetic polarity, it would be desirable to present a more complete view of the debate in the field about this issue. For example, Ruiz et al, (doi.org/10.1371/journal.pbio.3001497) recently provided structural arguments for the NTD being the face used for monomer addition without detecting the same intermediate form reported in this manuscript. How do their data and arguments differ from your findings? More generally, isn´t the fact that the NTD does not change substantially as FtsZ transitions from R to T already an argument for the NTD being the surface used for monomer addition?
    6. l. 74 "led us to propose a structural basis for the kinetic polarity of FtsZ, where transition of the NTD to the T-state conformation driven by GTP binding is sufficient to add a GTP-bound monomer to the bottom interface of the FtsZ filament." This statement suggests that GTP is necessary for the intermediate conformation but this is not supported by the data, as the GDP bound 7YSZ structure also has one monomer in the intermediate conformation. As far as I can tell, there is no structural evidence to suggest that the nucleotide gamma phosphate plays any role in the R-T transition. Even the role of the gamma phosphate in organizing the T3 loop in an assembly-conducive conformation seems to still be a controversial matter in the field. According to Matsui 2014 (doi.org/10.1074/jbc.M113.514901) "based on the results of the present study as well as on the structures deposited previously by other groups (PDB codes 2RHL, 2RHO, 2Q1X, and 2Q1Y) (43, 44), nucleotide exchange appears not to directly induce a structural change in the monomer, including the T3 loop."
    7. The experiments with the reciprocal cleft mutation in E. coli are not very informative as it is difficult to correlate the division defect in vivo with specific kinetic defects of the mutant FtsZ. The authors should have at least done a basic characterization of the E. coli mutant in vitro to demonstrate that it is altered in its CC alone. In fact, the dominant negative effect of the mutation in vivo is not something one expects from a poorly nucleating protein, which, if anything, should have a hard time poisoning the endogenous protein. The effect on ring compaction also suggests that the mutation must affect the protein in a broader way, perhaps including filament geometry. I would suggest that this part of the manuscript could be excluded without any loss for the SmFtsZ conclusions.

    Minor points

    1. l. 14 "CTD of the nucleotide-bound monomer cannot bind to the NTD-exposed end of the filament unless relative rotation of the domains leads to cleft opening." This is not accurate. There is no steric impediment to this reaction. Monomers in R conformation should be able to add to the NTD end of the filament as well, even if this is slower than the opposite reaction. The absence of growth from the NTD end is because the rate of addition/conformational change is slower than the rate of GTP hydrolysis.
    2. The comparison between the 7YOP (B) structure and the S. aureus 3WGN structure to show the effect of the gamma phosphate on T3 loop structure should be presented in a single figure, instead of being split between Fig. 4 and Fig. S2, and preferably using similar poses of the two structures. In the current state, it is quite hard to visualize the similarities mentioned by the authors.
    3. In contrast to what´s in the main text (l. 130), the chain with continuous density in Figure 2 is assigned as B, not A. Please clarify which is correct.
    4. l. 271 pBAD is the plasmid name, not the promoter. The promoter is PBAD(subscript).

    Significance

    This is a good quality manuscript overall, but which could still be improved by the suggestions below. In terms of significance, it provides new data to support current models for FtsZ assembly mechanism but no major new insights. The findings are interesting for a more specialized audience.

  5. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    Summary

    This is a study of cell division protein SmFtsZ from Spiroplasma melliferum, a cell wall-less Mollicutes bacterium where FtsZ may provide the primary force for division. Using X-ray crystallography, biochemical and microbiological experiments, the authors provide insight into how FtsZ's relaxed (R) to tense (T) conformational switching and its GTPase activity explain the kinetic polarity of FtsZ filament treadmilling. They propose: 1) an intermediate R/T state of FtsZ that facilitates preferential binding of N-terminal domain (NTD) of a monomer to C-terminal domain (CTD) of the terminal subunit at the filament bottom end; 2) that R to T switching is the rate-limiting step of FtsZ polymerization; 3) a T3 loop mechanism for GTP gamma phosphate triggering FtsZ polymerization.

    All comments and criticisms below are made for the sake of this interesting study.

    General Comments

    The study is well thought, carefully executed, and the manuscript is well written. However, the first conclusion is not convincing, because it is based on a misleading analysis; and the third conclusion is complicated by the use of an unqualified analog of GTP. SmFtsZ crystallizes as a dimer with the NTD and CTD domains swapped and a NTD-NTD contact. Mechanistic conclusions drawn from this unusual structural context are largely speculative, as they may not hold for normal FtsZ assembly. The NTD structure changes really very little between R-FtsZ and T-FtsZ, so that it is probably incorrect to define a T-NTD/R-CTD intermediate conformation from the guanine ring angle, as will be reasoned below. In addition, the GTP analog GMPPNP employed to investigate the effects of the gamma phosphate is not demonstrated to promote FtsZ assembly as GTP does; in fact, its beta-gamma phosphate geometry in the SmFtsZ structure is clearly different from GTP in other FtsZ structures. This raises the concern that GMPPNP may be not a bona fide functional analog of GTP for FtsZ.

    The authors should moderate the first and third claims, keeping speculations for discussion. Additional experiments required to assess the activity of GMPPNP inducing FtsZ assembly should be reported, even if the result was negative. Authors may consider partially refocusing the manuscript, including the title, towards the mechanism of the R to T transition, with Phe224Met modulating the opening of the cleft between NTD and CTD. Careful discussion of the structural basis of the kinetic polarity of FtsZ filaments in the light of previous and current results should be fine. In addition, SmFtsZ is now the third FtsZ that has been crystallized as a domain swapped dimer, suggesting a tendency for intramolecular dissociation of NTD and CTD with potential mechanistic implications, as the authors point out by the attractive end of the discussion.

    Specific comments (major and minor)

    Line 37 should read "...connected via H7 helix (15), with divergent C-terminal extensions".

    Line 55 Please note that the kinetic polarity of FtsZ has been deduced from mutational analysis (rather than observed as in the case of microtubules)

    Line 75 ""..transition of the NTD to the T-state conformation driven by GTP binding is sufficient..." This sentence appears conceptually wrong, because the R of T conformation of FtsZ is deemed independent of GTP or GDP binding in the literature (for example, Ref 23)

    Line 89 should read " in the presence of GTP and Mg2+ and not with GDP and Mg2+"

    Line 90-Figure 1A. The greyish gel electrophoresis image and those in SI require improving staining or photos. Standard Coomassie staining typically gives less background and better contrast.

    Line 90. Were the SmFtsZ filaments single or multiple in EM?

    Line 92. "...other characterized bacterial FtsZs" Some references should be cited

    Line 107 suggesting -> indicating

    Lines 120-122 " a truncated construct....SmFtsZdeltaCt showed similar GTPase activity as the wild type" is repeated from lines 110-111 above

    Line 124. Why the choice of GMPPNP, rather than GTP or GMPCPP?. Have SmFtsZ structures with GTP or GMPCPP been attempted?

    Line 124. It would be helpful to the reader to explain here that structure 7YSZ has two GDP-bound chains whereas structure 7YOP has GDP in chain A and GMPPNP in chain B.

    Lines 131 and 133. The names of chain A and chain B are swapped in the text Figure 2A-D. Consider enhancing the nucleotide tracing for easier visualization

    Line 141 and Figure 2F. Why change from the refraction detector in panel E to the absorbance detector in panel F? Importantly, how to know whether the shoulder corresponds to a dimer or to an extended monomer, and was the column calibrated?. In any case, do extended monomers and domain swapped dimers exist in solution? Additional crosslinking experiments, and analytical ultracentrifugation if available, could provide interesting results, although this is not a strict requirement for this manuscript.

    Lines 155-157 and Figure 3. The "GTP-bound T-state" 3WGN structure is not GTP but GTP-gamma S-bound, which makes a difference. 3WGM is GTP-bound SaFtsZ, although with a truncated loop T7. There is an unnecessary mix of FtsZs from different species in structures 2RHL and 3VOA; using instead 5H5G-molecule A (T-state, GDP) and 5H5G-molecule B (R-state, GDP) would simplify the structures employed for comparison in Figure 3 to a single species, SaFtsZ. In fact, 5H5G is employed as a reference in Figure 3C, although the distinction between 5H5G molecules A and B is not mentioned.

    Lines 157-160. The guanine ring angle depends on a stacking interaction with Phe183 form helix H7, which shifts in known FtsZ R and T structures. But this part of the structure is actually missing from the so called "T-state GDP" and "T-state GTP" SmFtsZ swapped domain structures. Instead, the guanine ring interacts with the main chain carbonyl of Phe137, an interaction which is not observed in the standard R or T FtsZ structures employed for comparison. This makes using the guanine ring angle alone misleading for conformational classification of SmFtsZ. In addition, both SmFtsZ "T-state" structures show a R-like Arg29 disengaged from interacting with the guanine (Figure 3), contrary to the interaction observed in the FtsZ T conformation. The overall conformation of the SmFtsZ structures does correspond to R-FtsZ. However, the swapped domain context of the SmFtsZ structure hampers meaningful comparisons with other FtsZ structures at a detailed local level around the guanine ring.

    Lines 175-177. "We concluded that in B chain the nucleotide-bound NTD is in T-state...". Importantly, the structure of the NTD of FtsZ, not including helix H7, is known to be very similar in the R and T conformations; differences are the position of helix H7, the position of the CTD relative to the NTD and the opening of the interdomain cleft (refs 23 and 24). The guanine ring angle is clearly related to the H7-Phe183 shift. Therefore, distinguishing R and T-conformations of the NTD in FtsZs and in SmFtsZ in particular seems unsupported by experimental data.

    Lines 197-199. Checking known FtsZ structures shows that Gly71 in loop T3 can be flipped out or in with GDP in both R and T conformation, whereas it is out with GTP or its analogs, making room for the gamma phosphate. It is interesting that the authors now observe this change with SmFtsZ, comparing the structures of GDP-bound and GMPPNP-bound protein. However, they should analyze and mention the precedents in the PDB, not only the GTP-gamma-S-bound 3WGN, and draw their conclusion very carefully due to the swapped domain context. There are known interactions made by the nucleotide gamma phosphate (PDB 3WGM) and one analog (PDB 7OHK) across the association interface in FtsZ filaments that explain FtsZ polymerization. In addition, is loop T3 really stabilized by the gamma phosphate of by filament formation?

    Lines 202-210. Tyr145 is not part of loop T5 but of helix H5. The observed interplay between loop T3 Pro73 and H5 Tyr145 is an attractive feature (apparently reminiscent of the tubulin T3-T5 story, but see Discussion). Please indicate if this has not been pointed out before in other FtsZs with the residue corresponding toTyr145, and consider analyzing existing FtsZ structures for T3-H5/T5 cross talk in different nucleotide states.

    Lines 212-324. The last three sections of Results convincingly demonstrate how residue 224 Phe/Met in the cleft between CTD and NTD modulates SmFtsZ assembly, EcFtsZ assembly, and E. coli cell division. In addition to this study, is it known whether SmFtsZ can replace EcFtsZ for E. coli cell division?

    Line 220 and Figure S3A. Please explain the color code in this Figure.

    Line 243. How can it be proposed that SmFtsZF224M could not be crystallized with GMPPNP probably due to efficient filament formation, if the activity of GMPPNP inducing filament formation has not been documented?

    Figure 6 panel F. The NeonGreen Z-ring microscopy images need enlargement to be properly appreciated.

    Discussion Line 342. Please notice that loop T3 is not always disordered with GDP. The proposal lacks an analysis of other FtsZ structures, in addition to 3WGN, and ignores intermolecular interactions of the nucleotide gamma phosphate and the coordinated Mg2+ ion (Matsui et al, 2014 J Biol Chem; Ruiz et al, 2022 PLoS Biol).

    Discussion Lines 356-370. The similarities to the classical GTP/GDP-dependent T3-T5 cross talk in the tubulin-RB3 complex (reviewed in Ref 27) is appealing, but notice that this was curved R-state tubulin with an accessory protein. But maybe the nucleotide dependent T3-T5 cross talk does not take place in T-tubulin from cryoEM microtubule structures with GDP and GTP (LaFrance et al and Nogales 2022 PNAS)?. And the authors should carefully check the tubulin T3 and T5 loop GDP/GTP-dependent conformations in the recently available cryoEM structures of free tubulin heterodimers (R-state) bound to GDP (PDB 7QUC) and GTP (PDB 7QUD) without any accessory proteins, which differ from the classical view.

    Discussion Lines 371-379. It should be noticed that a simpler interpretation of the results is that SmFtsZ is in the R-state, with R-CTD and R-H7, whereas the NTD is practically the same in both R and T states, as for other FtsZs (Ref 23). The T-like guanine angle may result from anomalous interactions of the swapped domains in SmFtsZ.

    Discussion Lines 381-384. There is really no need to postulate a NTD transition from R- to T-state in order to propose a kinetic polarity for the FtsZ filament from structure. In fact, having the NTD conformation constant results in a monomer top interface that is pre-formed for association and with the help of GTP should bind to the filament bottom subunit, as already proposed in Ref 35.

    Referees cross-commenting

    In addition to the concerns shared by the reviewers, especially those related to the existance or the role of distinct R and T conformations of the NTD of FtsZ, as welll as the individual reviewer concerns, we would like to highlight the relevance of:

    The comment of reviewer 1, requiring more information on the biological role of FtsZ in cell division of Spiroplasma and whether it forms a ring.

    Comment 2 of reviewer 3, requiring time-dependance of SmFtsZ polymerization and GTPase data, which are essential for properly analyzing the GTPase activity.

    Significance

    This interesting work, if successfully revised, will provide valuable insight into how the FtsZ polymerization switch and the nucleotide binding loops work for assembly of polar filaments, employing FtsZ from a wall-less bacterium. Please see the comments above for the existing literature context of the manuscript. This paper will be possibly suitable for a general biological audience, in addition to microbiologists and cytoskeletal researchers.

    This review has been prepared by biochemistry and structural biology experts familiar with FtsZ, hoping that it may be useful to the authors.

  6. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Summary: This paper reports the biochemical and structural characterization of FtsZ from a wall-less bacterial organism, Spiroplasma melliferum. From the analysis of the crystal structures (and comparison to known structures) the authors propose a model to explain the kinetic polarity of FtsZ treadmilling that was derived from a mostly genetic analysis (ref 26). In that model FtsZ with GTP bound adds to the bottom end of a filament with the C-terminal domain then shifting to the T-state. The status of the N-ter (whether in the T or R-state was not considered). Here the authors have proposed that they have captured an intermediate with the N-ter in the T-state and the C-ter in the R-state. It is proposed that this form adds to the bottom of a filament with the C-ter now adopting the T-state. I am not convinced this model is supported by the data as it is not clear when the N-ter domain switches to the T-state (before or after addition the end of a filament).

    Significance

    Note: the authors define the N-ter being in the T-state vs R-state based on the orientation of the guanine ring. The C-ter domain is in the T vs R-state based on whether the cleft is open or closed respectively.

    The basis of the authors' model comes mostly from the analysis of the crystal structure of FtsZ from the wall-less bacterial that was obtained in this study. The crystal structure revealed an unusual swapped dimer. Although in solution this FtsZ is a monomer, it crystallized as a swapped dimer indicating that during crystallization FtsZ domains came apart and reassociated with the opposite domains from another monomer. Careful analysis reveals that in one monomer the N-ter domain is in the T-state whereas in the other the N-ter domain is in the R-state (independent of nucleotide; GDP or GMPCPP). Although the N-ter domain is in the T-state in both monomers there are some differences - with GMPCPP the T3 loop is ordered whereas it is disordered with GDP. Also, they propose that the orientation of Gly71 is such in the GTP state that it favors interaction with the bottom end of a filament.

    Is it known whether FtsZ assembles into a Z ring and is required for cell division in this organism?

    In the dimer both C-terminal domains are in the R-state. From this the authors propose that the one monomer in the dimer is in the R-state whereas the other is in transition state (T-for N-ter and R-for C-terminal domain).

    The authors analyze sequences of FtsZ from different bacteria and notice that position 242 is a Phe in their organism whereas it is a Met in other bacteria. They wonder whether this residue influences the C-ter transitioning to the T-state so they swap residues - putting a Met in Sm and a Phe in Ecoli at this position. Interestingly, they notice that Sm-FtsN-met results in increased GTPase activity but longer filaments - this seems contradictory as higher GTPase is usually associated with shorter filaments - e.g. increased Mg slows GTPase activity and increased filament length and bundling. The Ec-FtsZ-Phe mutant displays increased cell length but not sure this can be ascribed to an effect on the GTPase activity.

    Overall, the work is well done and it comes to interpretation and whether the data support the model. The emphasis is on the N-ter getting to the T-state, but I am not sure that is the important step. It seems to me that rate-limiting step in FtsZ assembly is the C-ter getting into the T-state, which happens when a subunit is added to the end of a filament. Obviously the N-ter has to get to the T-state as well but how that happens is not clear. Presumably, it happens as a GTP-bound monomer in the R-state adds to the end of a filament resulting in the N-ter adopting the T-state followed by the C-ter adopting the T-state. In other words the T-state is only achieved by addition of a subunit to the end of the filament.

  7. Version published to 10.1101/2022.10.13.512043v2 on bioRxiv
  8. Version published to 10.1101/2022.10.13.512043v1 on bioRxiv