Crosslinking by ZapD drives the assembly of short FtsZ filaments into toroidal structures in solution
- Department of Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, Germany
- Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Germany
- Exzellenzcluster ORIGINS, Germany
- Molecular Interactions Facility, Centro de Investigaciones Biológicas Margarita Salas, Consejo Superior de Investigaciones Científicas (CSIC), Spain
- Cryo-EM Facility, Max Planck Institute of Biochemistry, Germany
- Centro de Investigaciones Biológicas Margarita Salas, Consejo Superior de Investigaciones Científicas (CSIC), Spain
- Helmholtz Pioneer Campus, Helmholtz Munich, Germany
- Department of Chemistry, Technical University of Munich, Germany
Figures
Figure 1 with 5 supplements
ZapD binds FtsZ and promotes filament bundling.
(a) Scheme of the FtsZ protein and its interaction with ZapD. E. coli FtsZ (blue) monomers in solution oligomerize depending on the buffer conditions. Upon GTP binding, FtsZ homopolymerizes directionally, assembling single-stranded filaments. ZapD is a robust dimer (magenta) that interacts directly with FtsZ, crosslinking filaments by promoting lateral interactions between them. Although the molecular mechanism is still unclear, the current hypothesis of interaction assumes dimers of ZapD crosslinking two FtsZ filaments through the CCTP of FtsZ, expecting at around 1:1 (FtsZ:ZapD) molar ratio in a homogeneous bundle (1 dimer of ZapD connected to 2 monomers of FtsZ). According to this model, the orientation of the FtsZ filaments could be parallel or antiparallel, allowing the growth and treadmilling of the filaments. However, the mechanism of assembly of dynamic high-order structures is still unknown. (b) Turbidity assays measuring the absorbance at 350 nm of samples containing 5 µM FtsZ and increasing concentrations of ZapD. The turbidity of the sample was measured 5 min after the addition of 1 mM GTP at working buffer (50 mM KCl, 50 mM Tris-Cl, 5 mM MgCl2, pH 7). FtsZ polymers do not show a significant turbidity at this wavelength; therefore, the signal at 350 nm corresponds to the presence of large FtsZ macrostructures and bundles. The mean value and SD of >3 independent replicates are plotted in the graph. (c) GTPase activity of FtsZ after the addition of 1 mM GTP in the presence of increasing concentrations of ZapD at working conditions (50 mM KCl, 50 mM Tris-Cl, 5 mM MgCl2, pH 7). The mean value and SD plotted in the graph are the result of 3 independent replicates. The GTPase activity was measured as a result of the Pi released from GTP consumption. The units are mol GTP consumed per mol FtsZ per min.
Figure 1—figure supplement 1
Characterization of the ZapD dimer and ZapD-FtsZ-GDP interaction by analytical ultracentrifugation (AUC).
(a) Sedimentation coefficient distribution, c(s), obtained by sedimentation velocity (SV) with (I) ZapD at different protein concentrations at working conditions (50 mM KCl, 50 mM Tris-Cl, 5 mM MgCl2, pH 7). More than 95% of the sample sedimented as single species with an experimental sedimentation coefficient of 3.5 S once corrected to standard conditions (s20, w: 3.6 S±0.1). It matched with the expected value for ZapD dimers. (II) Sedimentation coefficient distribution, c(s), obtained by SV of FtsZ at 15 µM at working conditions (50 mM KCl, 50 mM Tris-Cl, 5 mM MgCl2, pH 7). (III) Sedimentation coefficient distribution, c(s), obtained by SV of the interaction of 15 µM FtsZ and increasing concentrations of ZapD in physiological glutamate-acetate buffer. There is an axis break at 1.6 to highlight protein complexes. (IV) Represents the global multi-wavelength (280 and 250 nm) analysis of the sedimentation coefficient distribution for FtsZ-ZapD complexes obtained by SV and decomposition into component sedimentation coefficient distributions ck(s) for FtsZ (solid trace) and ZapD (dashed trace) using FtsZ:ZapD initial ratio of 1:4. Numbers over the peaks correspond to the FtsZ:ZapD molar stoichiometry observed for each complex. (b) Concentration gradient obtained by sedimentation equilibrium (SE) of ZapD at 10 µM. Experimental data (empty circles) are shown together with the best-fit analysis corresponding to ZapD dimer (solid line), monomer (dashed line), and trimer (dotted line). The lower plot represents the residuals of the fitting of the experimental data to the dimeric model. ZapD was shown as a stable dimer, showing a buoyant mass of 14,415 Da, corresponding to a molecular mass of 56,200±400 Da, considering the partial specific volume calculated from the ZapD amino acid sequence.
Figure 1—figure supplement 2
Biochemical characterization of the ZapD-FtsZ-GDP interaction by fluorescence correlation microscopy (FCS) and fluorescence anisotropy.
(a) Interaction curve of FtsZ-GDP and ZapD by fluorescence anisotropy. The fluorescence anisotropy measurement of 5 µM ZapD supplemented with 150 nM ZapD-ATTO 647 N at increasing concentrations of FtsZ demonstrated a direct interaction between both proteins with an apparent Kd of 10.26±1.07 (SE). Samples were in working conditions (50 mM KCl, 50 mM Tris-Cl, 5 mM MgCl2, pH 7) and the error bars represent the standard deviation among 3 independent samples. (b) FCS analysis of increasing concentrations of FtsZ-GDP with 5 µM of ZapD supplemented with 100 nM ZapD-ATTO 647 N in working conditions. The FCS analysis demonstrated an increasing diffusion time of ZapD along with the FtsZ concentration as a result of higher proportion of ZapD bound to FtsZ. Negative controls were measured with either FtsZ or ZapD alone. The plotted line is only to guide the eye. Each condition was measured in three independent samples.
Figure 1—figure supplement 3
Biochemical characterization of the ZapD and FtsZ bundles by turbidity at 350 nm.
The characterization of the FtsZ bundles resulting from the interaction with ZapD was made by measuring the turbidity of the samples at 350 nm in a plate reader. The mean value and standard deviation are the result of 3–6 independent samples. The signal from the blanks was collected before polymerization of FtsZ and subtracted for further measurements. Control of ZapD and FtsZ polymers independently did not show any significant difference with the blank after GTP addition. The working buffer was used unless it is specified in the legend (50 mM KCl, 50 mM Tris-Cl, 5 mM MgCl2, and pH 7). The absorbance was measured every 5 min for 100 min. (a) FtsZ bundling at different concentrations of FtsZ with 5 µM ZapD and 1 mM GTP in working conditions. In this case, the signal from FtsZ bundles was normalized to the maximum absorbance achieved (7.5 µM FtsZ) to facilitate the interpretation of the results. The signal plotted corresponds to 5 min after the addition of GTP. There was a clear decrease in the signal obtained at high concentration of FtsZ due to the low protein ratio ZapD/FtsZ. (b) Assembly of FtsZ bundles at different ionic strength conditions using KCl. The concentration of FtsZ and ZapD was 5 µM for both proteins, adding 1 mM GTP in the working buffer supplemented with different concentrations of KCl (50–500 mM KCl). Higher ionic strength in the buffer reduced the amount or size of bundles formed in solution, as the FtsZ-ZapD interaction is decreased. (c) Effect of pH in the FtsZ bundling process. The protein concentration used was 5 µM of ZapD and FtsZ with the addition of 1 mM GTP in the working buffer at different pH (6.5–8 pH). Lower pH enhanced the amount of FtsZ bundles promoted by ZapD, as lateral FtsZ-FtsZ interactions are promoted at lower pH. (d) Analysis of FtsZ bundling over time when ZapD was mixed with FtsZ prior or after the GTP addition and polymerization. The concentration of FtsZ and ZapD was 5 µM and 1 mM GTP in working buffer, respectively. ZapD mixed with FtsZ before or after GTP addition did not show significant difference in the formation of FtsZ bundles. (e) Measurement of turbidity over time of 5 µM FtsZ in the presence of ZapD at increasing concentrations. Both proteins were mixed prior to the addition of 1 mM GTP. A decay in the signal can be observed over time, highlighting the dynamic nature of the FtsZ bundles. After 100 min, an extra 1 mM GTP was added and FtsZ bundles were reassembled, showing lower increase of the signal due to a higher concentration of GDP in the samples, likely shifting the reaction. The addition of 1 mM GTP was done twice after 100 min.
Figure 1—figure supplement 4
Ionic strength in the buffer lowers the effect of ZapD over FtsZ GTPase activity.
GTPase activity of FtsZ in the presence of equimolar concentration of ZapD (5 µM) after the addition of 1 mM GTP at different ionic strength conditions (50–500 mM KCl, 50 mM Tris-Cl, 5 mM MgCl2, pH 7). The mean value and SD of the FtsZ GTPase activity correspond to 3 independent replicates. The GTPase activity was measured as a result of the Pi released from GTP consumption. The units are mol GTP consumed per mol FtsZ per minute. Moderate salt concentrations (100–200 mM) usually support optimal FtsZ assembly and GTP hydrolysis.
Figure 1—figure supplement 5
ZapD binding to FtsZ polymers via sedimentation assays.
(a) Analytical sedimentation velocity (SV) assay. This table summarizes the binding of ZapD to FtsZ polymers, determined by the SV of FtsZ at 5 µM and increasing concentrations of ZapD (1–30 µM). The experiments were done at working conditions. (b, c) Pelleting assays using a preparative centrifuge. Samples of FtsZ (5 µM) and increasing concentrations of ZapD (1–30 µM) were sedimented at 10,000 rpm, and the pellets and supernatants were subjected to SDS-PAGE (see panel c). The relative intensities in the electrophoresis allowed a qualitative estimation of the concentrations of FtsZ in the supernatant and pellet. (b) shows the estimation of FtsZ in the pellet and supernatant. The protein concentrations and buffer conditions used in this assay were the same as those in the analytical SV assay. (c) The samples loaded in the SDS gel from left to right include FtsZ at 5 µM, ZapD at 10 µM, and FtsZ in the presence of 1, 5, 10, or 30 µM ZapD. The molecular weight markers from top to bottom are 50, 37, 25, 20, and 15 kDa. ‘C’ denotes the control sample without centrifugation, while ‘S’ represents the supernatant, and ‘P’ indicates the pellet.
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Figure 1—figure supplement 5—source data 1
Raw images of the SDS-PAGE gels used in Figure 1—figure supplement 5 and for quantification, corresponds to the raw SDS-PAGE gels.
- https://cdn.elifesciences.org/articles/95557/elife-95557-fig1-figsupp5-data1-v1.zip
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Figure 1—figure supplement 5—source data 2
Raw images of the SDS-PAGE gels used in Figure 1—figure supplement 5 and for quantification, includes the labelled images.
- https://cdn.elifesciences.org/articles/95557/elife-95557-fig1-figsupp5-data2-v1.zip
Figure 2 with 1 supplement
ZapD promotes the formation of FtsZ toroids.
(a) Cryo-electron microscopy (cryo-EM) micrographs of FtsZ filaments (FtsZ-GTP form) (left) and ZapD protein (right) at 10 µM under working conditions (50 mM KCl, 50 mM Tris-Cl, 5 mM MgCl2, pH 7). Scale bars are 100 nm. (b) Cryo-EM images of FtsZ (10 µM) in the presence of equimolar concentrations of ZapD (ratio 1:1) and 1 mM GTP in working conditions. Cryo-EM grids were plunge-frozen 2 min after GTP addition to favor the assembly of FtsZ and ZapD structures. The proteins were mixed before the polymerization was triggered by GTP addition. Scale bars are 250 nm. (c) Micrograph of an individual FtsZ toroid found under the same conditions as in (b). Close-up view of an area within the toroid is framed by a dotted black line, revealing the large amount of FtsZ filaments that form its structure. (d) Distribution of the outer diameter of the FtsZ toroid. Each toroid was measured perpendicularly in the shortest and longest axis to ensure the reliability of the measurement. The mean value and standard deviation are shown in the graph. (e) Distribution of toroidal thickness. It was measured as the result of the difference between the outer and inner diameter of each toroid. The mean value and standard deviation are shown in the graph.
Figure 2—figure supplement 1
FtsZ toroids and bundles formed at different protein ratios.
(a) Cryo-electron microscopy (cryo-EM) images of FtsZ bundles and toroids promoted by interaction with ZapD at different protein ratios. From top to bottom, the protein ratios used in the samples were 1:1, 1:2, 1:4, 1:6 for FtsZ at 10 µM and ZapD concentrations ranging from 10 to 60 µM. These ratios do not refer to the stoichiometry of the proteins forming the structure. Samples were plunge-frozen 2 min after the addition of 1 mM GTP. In these samples, similar toroidal structures and bundles were observed regardless of the protein ratio, including spirals and curved bundles. Scale bars are 250 nm. (b) Distribution of toroid heights measured from cryo-electron tomography (cryo-ET) data. Cross-sections of the toroids in the XZ plane were used to measure the height of each toroid. Distance measurements were taken from each toroid in the middle of the bundle at the four cardinal points to assure the reliability of the results. The mean value and SD are shown in the graph. (c) Circularity of FtsZ toroids. For each toroid, the shortest and largest distances of the outer and inner diameter were measured from the cryo-EM images. The division between the shortest and longest diameter provided the circularity of the FtsZ toroids. The mean value and SD are shown in the graph. The circularity of the inner and outer diameter is significantly different (t-test t(4.2), p-value<0.001).
Figure 3 with 4 supplements
Three-dimensional (3D) structure of FtsZ toroids revealed by cryo-electron tomography (cryo-ET).
(a) Representative tomographic slice of an FtsZ toroid resulting from the interaction of FtsZ with ZapD. The image is the average of five 0.86-nm-thick tomographic slices (total thickness of 4.31 nm) of the reconstructed tomogram around the equatorial plane of a single FtsZ toroid. The concentrations of FtsZ and ZapD were 10 µM, and 1 mM of GTP was added to initiate polymerization under working conditions (50 mM KCl, 50 mM Tris-Cl, 5 mM MgCl2, pH 7). (b) Close-up views of the toroidal structure show the alignment of the FtsZ filaments forming the toroid. Red arrows indicate the presence of connections between filaments. (c) The tomographic slice in the XZ plane (left) shows the cross-section corresponding to the area marked by the white dotted line in b This image is the average of nine tomographic slices (total thickness of 7.74 nm) from the denoised tomogram. The isosurface of the cross-section (right) shows the vertical alignment and stacking of the FtsZ filaments within the toroid. This suggests that the interaction between FtsZ filaments and ZapD is mainly along the Z direction. FtsZ filaments are represented in blue. (d) Isosurface of the FtsZ toroid shown in a. It was extracted from the reconstruction of the denoised tomographic volume and positioned in different views to facilitate its visualization: (top) front view, (middle) side view, and (bottom) lateral view. The toroid has a diameter of ~552 nm and a height of ~92 nm. (e) A close-up view of the segmented toroidal structure. It shows the complex internal organization of filaments assembling the toroid. It corresponds to a zone within the toroid shown in b on the right. Close-up views of the isosurface show different connections between filaments. The segmentation shown has a width of 136 nm × 101 nm and a height of 64 nm.
Figure 3—figure supplement 1
FtsZ structures found at low and high ZapD concentrations.
Representative tomographic slices of FtsZ structures and bundles promoted by ZapD at different protein ratios under working conditions. The images are an average of five 0.86 nm slices from the reconstructed tomographic volume (total thickness of 4.31 nm) to enhance the signal-to-noise ratio of the images. Scale bars are 250 nm unless they are labeled with 100 nm. Samples were plunge-frozen 2 min after triggering polymerization. (a) Equimolar concentrations of FtsZ and ZapD (10 µm) in the sample formed toroids and bundles after 1 mM GTP addition. (b) High concentration of ZapD (60 µm) promoted the formation of straight FtsZ bundles, although they coexisted with scarce amounts of FtsZ toroids and double filaments showing striated patterns.
Figure 3—figure supplement 2
Segmentation of an FtsZ toroid imaged using cryo-electron tomography (cryo-ET).
(a) Tomographic slice of a toroidal FtsZ structure. The image is an average of five 0.86 nm slices from the reconstructed tomographic volume (total thickness of 4.31 nm). FtsZ and ZapD were added to the sample at equimolar concentrations (10 µm) before the addition of 1 mM GTP. The white dotted line represents the localization of the cross-section shown in (e), while the black dotted square shows the area of a close-up view shown below the image. The scale bar of the zoomed area is 50 nm. (b) Corresponding segmentation of the toroid shown in a The isosurface of the toroid was extracted from the denoised tomographic volume and positioned in different views: front (top), side (middle), and lateral (bottom). The curved filaments observed in the upper part of the toroid correspond to a bundle that was located in the upper layer of the toroid. It has been erased to make the toroid easier to see. The toroid has a diameter of ~581 nm and a height of ~119 nm. (c) Segmentation of the zoomed area shown in a. Top view of the isosurface from the toroidal structure. (d) Cross-section of the toroid in the XZ plane (left; plane indicated by the white dotted line in a) and corresponding isosurface (right). The tomographic slice is the average of nine tomographic slices. (e) Different views of the isosurface of the toroidal structure. The network of filaments shown corresponds to the image shown in (c) in different views. The segmentation shown has a width of 116 nm × 150 nm and a height of 82 nm.
Figure 3—figure supplement 3
Cross-section of the toroid showing the elongated structures in the Z-axis.
(a) Tomographic slice of a toroid. The image is an average of five 0.86 nm slices from the reconstructed tomographic volume (total thickness of 4.31 nm). The tomographic slice in the XZ plane shows a cross-section of the toroid (middle). This image is the average of nine tomographic slices (total thickness of 7.74 nm) from the denoised tomogram. Corresponding isosurface of the same area (right). (b) Different views of the isosurface of a filament network extracted from a toroid. The red dotted lines indicate the area that was selected for the cross-sections. Black dotted squares show closer views of the connections between filaments in the Z-plane. The segmentation shown has a width of 28 nm × 37 nm and a height of 56 nm.
Figure 3—figure supplement 4
Segmentation of FtsZ filaments.
(a) Isosurface of single and double FtsZ filaments in the absence of ZapD extracted from the denoised tomographic volume. FtsZ filaments observed from cryo-electron tomography (cryo-ET) samples in the presence of 2 mM GTP. The single and double filaments were extracted from the tomographic slice shown to the left side of each example. Top and side views of the segmented filaments are shown. Scale bars represent 50 nm. (b) Comparison of isolated FtsZ filaments from the following samples: (top) absence of ZapD, (middle) a toroidal structure at equimolar concentrations of FtsZ and ZapD (10 µM), and (bottom) a straight bundle at high concentration of ZapD (60 µM). The protein ratios added to the sample in each case are shown in the left side of each example. Side and top views of the three segmented filaments are shown to the left and middle columns, while a cross-section of the filaments is shown in the right column. The cross-section corresponds to the dotted line shown at the side top view. Different shades of blue distinguish the three conditions analyzed. The presence of extra densities decorating the FtsZ filaments strongly suggests that these are ZapD proteins decorating and connecting the filaments. These densities generate an elongation in Z beyond the missing wedge, which is clearly visible.
Figure 4 with 2 supplements
FtsZ filaments are connected by putative ZapD crosslinkers to assemble the toroidal structure.
(a) Top (left, top), side (left, bottom), and lateral (right) views of the isosurface from a region within the toroidal structure shown in Figure 3a. The FtsZ filaments are colored in blue, while filament connections or putative ZapD proteins are labeled in magenta to facilitate interpretation of the results. Other putative ZapD proteins decorating the FtsZ filaments were not labeled in magenta because they were not forming any clear linkage between the filaments. The segmentation shown has a width of 73 nm × 101 nm and a height of 64 nm. (b–d) Various examples of filament connections by putative ZapD proteins within the toroid. Same color code as in (a). From left to right, the localization of the analyzed region, a close-up view of the structure of interest, different views of the crosslinkers, and a schematic illustrating the interpretation of the data. The schematic (right) shows the localization of ZapD proteins (magenta) and FtsZ filaments (blue). (b) Lateral connection of two FtsZ filaments by a putative ZapD dimer. In this example, the attachment of each globular density or putative ZapD monomer was bound to each filament, allowing for lateral binding. (c) Putative ZapD connections stabilizing two filaments by a lateral interaction. Additional ZapD decorations attached to only one of the filaments appear to be available for other filament connections. (d) Multiple ZapD proteins can connect to filaments and stabilize the interaction. First, the two upper filaments are connected vertically by several putative ZapDs. The lower filament connects vertically in an oblique angle to the nearest neighboring filament. In the upper part, additional decorations or putative ZapD proteins would be available to establish further interactions forming a three-dimensional (3D) mesh.
Figure 4—figure supplement 1
The FtsZ toroid is formed by short and discontinuous filaments crosslinked by ZapD.
(a) Image of the isosurface of the network of filaments shown in Figure 4a. A black marker (sphere) is positioned at each discontinuity or termination of FtsZ filaments (blue) within the toroid. Magenta markers were placed at each clear connection between FtsZ filaments to analyze their presence in areas close to discontinuities. A closer inspection of the image, framed by a dotted black line, shows the termination and interruption of some FtsZ filaments. The black marker has been replaced by a dotted circle to show the absence of filaments in this area. The images on the left and right are just different areas of the same network of filaments within the toroid. (b) Images on the left and in the middle represent the spatial three-dimensional (3D) localization of discontinuities (black) and connections (magenta) of FtsZ filaments in the analyzed area of the toroid shown in (Figure 4a) (blue filaments). A top and side view of the structure shows the localization of the discontinuities and connections. The overlap of the two (right) shows no significant colocalization or correlation between them. The segmentation shown has a width of 73 nm × 101 nm and a height of 64 nm.
Figure 4—figure supplement 2
FtsZ single and double filaments found at different FtsZ and ZapD ratios.
Cryo-electron microscopy (cryo-EM) images of single and double filaments found together with toroids and bundles in samples containing FtsZ and ZapD at different protein ratios in the sample. The FtsZ concentration was 10 µM, while ZapD was present at increasing concentrations (from top to bottom 0, 2.5, 5, 10, 20, 40, and 60 µM), mixed before the addition of 1 mM GTP. These ratios refer to the initial protein concentration added and not to the stoichiometry of the two proteins forming the structure. In the cryo-EM samples, only single and double filaments were found, as shown in samples from 1:0 to 1:1 protein ratio. In these filaments, we could not discard or confirm the presence of ZapD connecting the FtsZ filaments. They were not easily distinguished from double filaments formed in the absence of ZapD as a consequence of weak FtsZ-FtsZ lateral interactions. In the case of saturation of ZapD in samples containing 1:4 or 1:6, double FtsZ filaments and straight bundles with a characteristic striped pattern perpendicular to the FtsZ filaments were found. Scale bars are 100 nm.
Figure 5 with 4 supplements
Formation of straight FtsZ bundles is driven by high ZapD crosslinking from above.
(a) Representative tomographic slices of straight FtsZ bundles resulting from the interaction of FtsZ with high ZapD concentrations under working conditions (50 mM KCl, 50 mM Tris-Cl, 5 mM MgCl2, pH 7). The concentrations of FtsZ and ZapD were 10 µM and 60 µM, respectively, and 1 mM of GTP was added to trigger polymerization. The straight bundles were found only at high ZapD concentrations. The image is the average of five 0.86-nm-thick tomographic slices (total thickness of 4.31 nm) of the reconstructed tomogram. Scale bars are 100 nm. (b) Isosurface of the straight bundles from the denoised tomographic volume. FtsZ filaments are colored in blue and putative ZapD connections in magenta. Three different views (top [left] and side views [middle, right]) are shown. Straight bundles are organized in a regular organization. Multiple bonds between filaments are formed vertically by putative ZapDs vertically crosslinking two FtsZ filaments with a regular spacing of 4.5±0.5 nm between ZapD dimers. In addition, lateral connections were also found, connecting pairs of stabilized filaments to each other and eventually assembling the straight bundle. (c) Different views of one of the isolated filaments from the straight bundle. A side view of the filaments (middle) shows a spike-like structure regularly located at the top of the FtsZ filaments, connecting them vertically as observed in the top view (right). (d) Different close-up views of the filament structure shown in (c). In the cross-section of the structure (middle, top), it is clearly visible that the ZapD proteins connect the two filaments vertically and from above, forming a bridge over them. A schematic of the proposed interaction (right, bottom) shows the position of putative ZapD dimers in this structure.
Figure 5—figure supplement 1
Segmentation of straight bundles imaged by cryo-electron tomography (cryo-ET).
Segmentation of straight bundles resulting from the interaction of FtsZ with high concentrations of ZapD under working conditions (50 mM KCl, 50 mM Tris-Cl, 5 mM MgCl2, pH 7). The concentrations of FtsZ and ZapD were 10 and 60 µM, respectively. 1 mM of GTP was added to induce polymerization and bundle formation. (a, b) show the isosurface of two straight bundles from the denoised tomographic volume. The entire volume has been colored in blue without differentiating ZapD connections to facilitate the interpretation. (b) shows different views of a cropped area within a straight bundle. The segmentation shown in (a) has a width of 270 nm × 175 nm and a height of 72 nm. Segmentation in (b) has a width of 36 nm × 65 nm and 60 nm in height.
Figure 5—figure supplement 2
Distance between FtsZ filaments and ZapD-associated filaments.
(a) Distribution of the distance between two ZapD proteins in a straight bundle under ZapD saturation (1:6 protein ratio). The distance between two ZapD proteins was measured from tomographic images obtained in samples containing FtsZ (10 µM) in the presence of ZapD at high concentration (60 µM) in the presence of GTP. The distribution obtained fitted a normal distribution. The mean value and SD are shown in the graph. The regular distance found between the ZapD proteins connecting the filaments could indicate the presence of one ZapD dimer per FtsZ monomer forming the filament. (b) Distribution of the distance between two FtsZ filaments (5.9±0.8 nm) in the absence or presence of ZapD forming toroidal structures (1:1) (7.88±2.09 nm) or straight bundles (1:6) (7.6±1.5 nm). The distance between filaments was measured from the cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) images and plotted in the graph as a distribution of distances. The three distributions were obtained from images of >3 independent samples. The mean value and SD are shown in the graph for each condition. The presence of ZapD crosslinking filaments increases the distance between them compared to weak FtsZ-FtsZ interactions. However, no significant differences were found between equimolar and saturation levels of ZapD.
Figure 5—figure supplement 3
mZapD binds FtsZ-GDP and can promote bundling of FtsZ filaments.
(a) Sedimentation coefficient distribution, c(s), obtained by sedimentation velocity (SV) with mZapD at different concentrations under working conditions (50 mM KCl, 50 mM Tris-Cl, 5 mM MgCl2, pH 7). mZapD sedimented mostly as a single species with an experimental sedimentation coefficient of 3.5 S when corrected to standard conditions (s20, w: 3.6 S±0.1). However, species at around 2.5 S appear at ~10%, in contrast to ZapD, demonstrating the presence of monomers and confirming the weakening of the dimerization interface. (b) Molecular interaction between FtsZ-GDP and ZapD or ZapD mutant (mZapD) measured by fluorescence anisotropy and compared with the results shown in Figure 1—figure supplement 2a for ZapD and FtsZ-GDP. 5 µM of mZapD was supplemented with 150 nM of mZapD-ATTO 647 N and FtsZ in increasing concentrations. mZapD (red) also binds FtsZ-GDP with an apparent Kd of 12.42±5.45 (SE). The X-axis is expressed in log units. The mean value and standard deviation are the result of 3 independent samples. (c) Fluorescence correlation spectroscopy (FCS) analysis of increasing concentrations of FtsZ-GDP with 5 µM of ZapD or mZapD supplemented with 100 nM ZapD or mZapD chemically labeled with ATTO 647 N. The plot corresponding to ZapD was the same as shown in Figure 1—figure supplement 2b and it is included here to facilitate the interpretation of the results. The X-axis is expressed in log units. The diffusion curve showed an increasing diffusion time of mZapD with increasing FtsZ concentration, supporting a direct interaction of this protein with FtsZ-GDP. The plotted line is to guide the eye only. Each condition was measured in three independent samples. (d) Turbidity signal of FtsZ and ZapD by measuring absorbance at 350 nm after 5 min of GTP addition. The concentration used was 5 µM FtsZ with increasing concentrations of ZapD (0–80 µM) and 1 mM GTP in working buffer. Data represents the mean and standard deviation of >3 independent samples. The signal shown for ZapD is the same as that shown in Figure 1b and it is included here to aid interpretation.
Figure 5—figure supplement 4
mZapD can bundle FtsZ without promoting toroids.
(a) Proposed schematic of the interaction mechanism between FtsZ-GTP and the mZapD protein. The mZapD protein is mutated at the dimerization site to weaken dimerization and favor monomerization of the protein. Thus, the interaction of dynamic FtsZ filaments with mZapD monomers is weakened, and the stabilization of toroidal structures or straight bundles is not possible, while dynamic bundles can still be formed. (b) Cryo-electron microscopy (cryo-EM) image of 10 µM mZapD. mZapD is homogeneously distributed, and it did not aggregate under our working conditions. The scale bar is 100 nm. (c) Representative cryo-EM images of FtsZ and mZapD at increasing concentrations of mZapD and 1 mM GTP under working conditions. The ratios shown on the left side of the images refer to the initial added protein concentration. At equimolar concentrations of FtsZ and ZapD (10 µM), no FtsZ bundles were found, and only single and double FtsZ filaments were observed (top). At higher concentrations of mZapD (20 and 60 µM, middle and bottom rows, respectively), FtsZ bundles were formed, although no toroids or straight bundles were observed in these samples. The absence of these structures suggested that a stable dimer is required to form them. Scale bars are 250 nm unless 100 or 50 nm labels are shown.
Figure 6
The amount of ZapD connections modulates the spatial organization of FtsZ filaments into higher-order structures.
(Top) Simplified scheme of the higher-order FtsZ structures formed in the presence of increasing concentrations of ZapD. These schemes do not consider all the possible interactions between ZapD and FtsZ. (Bottom) Cryo-electron microscopy (cryo-EM) images of the structures shown in the schemes. In the absence of ZapD, FtsZ filaments can interact laterally to form double filaments upon GTP binding. At low concentrations of ZapD, only a few ZapD-mediated bonds are formed, favoring the formation of small, curved bundles. At equimolar concentrations of ZapD and FtsZ, more ZapD-mediated bonds are formed, particularly from above the filaments, but also laterally and diagonally, allowing filament curvature and favoring the assembly of toroidal structures. At saturating ZapD concentrations, the filaments are straightened up by regular ZapD crosslinking vertically, resulting in the formation of straight bundles. Overall, the assembly of higher-order FtsZ structures depends on the number of vertical crosslinks through ZapD dimers. Some intermediate states are expected between the structures shown. Scale bars are 100 nm.
Videos
Video 1
Tomogram of an FtsZ toroidal structure promoted by ZapD shown in Figure 3a, followed by its segmentation.
FtsZ filaments are in blue. Successive rotations of the segmented volume allow us to visualize the structure of the toroid in three dimensions (3D).
Video 2
Isosurface of the toroid shown in Figure 2a.
FtsZ filaments are shown in blue and putative ZapD connections in magenta. A close-up view and rotations of the segmented volume show the filament meshwork and the connections by ZapD in three dimensions (3D).
Video 3
Tomogram of an FtsZ straight bundle formed at a high concentration of ZapD proteins shown in Figure 5b.
Successive segmentation of the tomogram with FtsZ filaments labeled in blue and putative ZapD connections in magenta. Rotations and close-up views help the interpretation of the data and show the three-dimensional (3D) structure of the straight bundle.
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/95557/elife-95557-mdarchecklist1-v1.pdf
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Source data 1
Raw data used to make the graphs shown in figures.
- https://cdn.elifesciences.org/articles/95557/elife-95557-data1-v1.xlsx
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Crosslinking by ZapD drives the assembly of short FtsZ filaments into toroidal structures in solution
eLife 13:RP95557.
https://doi.org/10.7554/eLife.95557.4
