Figures and data
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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 C-terminal region of FtsZ, expecting at around 2:1 (FtsZ:ZapD) molar ratio in an homogeneous bundle. 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 minutes 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 addition of 1 mM GTP in 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.
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ZapD promotes the formation of FtsZ toroids.
a 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.
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3D structure of FtsZ toroids revealed by cryo-ET.
a Representative micrograph of a FtsZ toroid resulting from the interaction of FtsZ with ZapD. The image is the average of five 6.4 nm thick tomographic slices (total thickness of 32 nm) of the reconstructed tomogram around the equatorial plane of a single FtsZ toroid. Scale bar is 150 nm. The concentrations of FtsZ and ZapD were 10 µM and 1 mM of GTP was added to trigger 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 FtsZ filaments forming the toroid. Red arrows indicate the presence of connections between filaments. c Cryo-ET image (left) shows the cross-section corresponding to the area located at the white dotted line in (b). This image is the average of nine tomographic slices from the denoised tomogram. The isosurface of the cross-section (right) shows the alignment and organization of the FtsZ filaments within the toroid. This suggests a regular distribution of filaments assembling the toroid. 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. 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 (d). Close-up views of the isosurface show different connections between filaments.
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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 an area within the toroidal structure shown in (Fig. 3a). The FtsZ filaments are colored blue, while filament connections or putative ZapD proteins are labelled magenta to facilitate interpretation of the results. Other putative ZapD proteins decorating the FtsZ filaments were not labelled in magenta because they were not forming any clear linkage between the filaments. b-d Different examples of connections between filaments by putative ZapD proteins within the toroid. Filaments are labelled in blue and putative ZapD in magenta to facilitate their interpretation. From left to right, the localization of the analyzed area, a close-up view of the structure of interest, different views of the crosslinkers and a schematic to illustrate the interpretation of the data. The schematic (right) represents 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 a lateral bond. c Putative ZapD connections stabilizing two filaments by a lateral interaction. Additional ZapD decorations attached only to one of the filaments appear to be available for other filament linkages. d Multiple ZapD proteins can connect to filaments and stabilize the interaction. First, the two upper filaments are connected to each other by lateral connections through several putative ZapD. The lower filament connects laterally with the two other filaments. In the upper part, additional decorations or putative ZapD proteins would be available to establish further interactions forming a three-dimensional mesh.
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Formation of straight FtsZ bundles is driven by high ZapD binding.
a Representative images 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 only found at high ZapD concentrations. The image is the average of five 6.4 nm thick tomographic slices (total thickness of 32 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 found in a very regular manner; putative ZapD proteins align and stack to stabilize two FtsZ filaments by crosslinking them. In addition, lateral connections were also found linking couple of stabilized filaments among them, ultimately 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 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, forming a bridge over them. A schematic of the suggested interaction (right, bottom) shows the location of putative ZapD dimer in this structure.
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Density of ZapD connections modulate the spatial organization of FtsZ filaments into high-order structures.
(Top) Scheme of the FtsZ FtsZ high-order structures formed in the presence of increasing ZapD concentrations. (Bottom) Cryo-EM images of the structures represented in the schemes. FtsZ filaments can interact laterally with each other, forming single and double filaments upon GTP binding. At low concentrations of ZapD, only a few lateral connections are formed and thus no bundles can be assembled. However, at higher ZapD concentrations, more ZapD-mediated bonds are established, favouring the formation of bundles. Higher ZapD binding densty promotes the assembly of toroidal structures. A further increment in the number of ZapD connections, the bundle is enriched on ZapD binding density, which straightens up the filaments allowing the formation of straight bundles. The assembly of FtsZ high-order structures is dependent on the number of lateral crosslinking through ZapD dimers and some intermediate states are expected between the structures shown. Scale bars are 100 nm.