Mechanism of FtsZ assembly dynamics revealed by filament structures in different nucleotide states

Treadmilling protein filaments perform essential cellular functions by growing from one end while shrinking from the other, driven by nucleotide hydrolysis. Bacterial cell division relies on the primitive tubulin homolog FtsZ, a target for antibiotic discovery that assembles into single treadmilling filaments that hydrolyse GTP at an active site formed upon subunit association. We determined high-resolution filament structures of FtsZ from the pathogen Staphylococcus aureus in complex with different nucleotide analogues and cations, including mimetics of the ground and transition states of catalysis. Together with mutational and biochemical analyses, our structures reveal interactions made by the GTP γ-phosphate and Mg2+ at the subunit interface, a K+ ion stabilizing loop T7 for co-catalysis, new roles of key residues at the active site and a nearby crosstalk area, and rearrangements of a dynamic water shell bridging adjacent subunits upon GTP hydrolysis. We propose a mechanistic model that integrates nucleotide hydrolysis signalling with assembly-associated conformational changes and filament treadmilling. Equivalent assembly mechanisms may apply to more complex tubulin and actin cytomotive filaments that share analogous features with FtsZ.

DivIVA is essential in Deinococcus radiodurans and its C terminal domain regulates new septum orientation during cell division

FtsZ assembly at mid cell position in rod shaped bacteria is regulated by gradient of MinCDE complex across the poles. In round shaped bacteria, which lack predefined poles and the next plane of cell division is perpendicular to previous plane, the determination of site for FtsZ assembly is intriguing. Deinococcus radiodurans a coccus shaped bacterium, is characterized for its extraordinary resistance to DNA damage. Here we report that DivIVA a putative component of Min system in this bacterium (drDivIVA) interacts with cognate cell division and genome segregation proteins. The deletion of full length drDivIVA was found to be indispensable while its C-terminal deletion (ΔdivIVAC) was dispensable but produced distinguishable phenotypes like slow growth, altered plane for new septum formation and angular septum. Both wild type and mutant showed FtsZ foci formation and their gamma radiation responses were nearly identical. But unlike in wild type, the FtsZ localization in mutant cells was found to be away from orthogonal axis with respect to plane of previous septum. Notably, DivIVA-RFP localizes to membrane during cell division and then perpendicular to previous plane of cell division. In trans expression of drDivIVA in ΔdivIVAC background could restore the wild type pattern of septum formation perpendicular to previous septum. These results suggested that DivIVA is an essential protein in D. radiodurans and the C-terminal domain that contributes to its interaction with MinC determines the plane of new septum formation, possibly by controlling MinC oscillation through orthogonal axis in the cells.

Modelling FtsZ nucleation, hydrolysis and treadmilling activity with Monte Carlo methods

ABSTRACTBacterial cell division is tightly coupled to the dynamic behavior of FtsZ, a tubulin homolog. Recent experimental work in vitro and in vivo has attributed FtsZ’s assembly dynamics to treadmilling, where subunits add to the bottom and dissociate from the top of protofilaments. However, the molecular mechanisms producing treadmilling have yet to be characterized and quantified. We have developed a Monte Carlo model for FtsZ assembly that explains treadmilling and assembly nucleation by the same mechanisms. A key element of the model is a conformational change from R (relaxed), which is highly favored for monomers, to T (tense), which is favored for subunits in a protofilament. This model was created in MATLAB. Kinetic parameters were converted to probabilities of execution during single, small time steps, and these were used to stochastically determine FtsZ dynamics. Our model is able to accurately describe the results of several in vitro and in vivo studies for a variety of FtsZ flavors. With standard conditions, the model FtsZ polymerized and produced protofilaments that treadmilled at 28 nm/s, hydrolyzed GTP at 2.8 to 4.2 GTP min-1 FtsZ-1, and had an average length of 25 to 54 subunits, all similar to experimental results. Adding a bottom capper resulted in shorter protofilaments and higher GTPase, similar to the effect of the known the bottom capper protein MciZ. The model could match nucleation kinetics of several flavors of FtsZ using the same parameters as treadmilling and varying only the R to T transition of monomers.SIGNIFICANCEFtsZ assembly dynamics are now known to be governed by treadmilling, where subunits add to the bottom and dissociate from the top of protofilaments. We have generated a Monte Carlo model of treadmilling based on (a) a conformational transition of FtsZ subunits between two states, and (b) stochastic GTP hydrolysis. Importantly, the nucleation of new protofilaments is explained by the same mechanisms as treadmilling. We have determined kinetic parameters that match a wide range of experimental data. The model is available to users for their own in silico experiments.

Nitric oxide disrupts bacterial cytokinesis by poisoning purine metabolism

Cytostasis is the most salient manifestation of the potent antimicrobial activity of nitric oxide (NO), yet the mechanism by which NO disrupts bacterial cell division is unknown. Here, we show that in respiring Escherichia coli, Salmonella, and Bacillus subtilis, NO arrests the first step in division, namely, the GTP-dependent assembly of the bacterial tubulin homolog FtsZ into a cytokinetic ring. FtsZ assembly fails in respiring cells because NO inactivates inosine 5′-monophosphate dehydrogenase in de novo purine nucleotide biosynthesis and quinol oxidases in the electron transport chain, leading to drastic depletion of nucleoside triphosphates, including the GTP needed for the polymerization of FtsZ. Despite inhibiting respiration and dissipating proton motive force, NO does not destroy Z ring formation and only modestly decreases nucleoside triphosphates in glycolytic cells, which obtain much of their ATP by substrate-level phosphorylation and overexpress inosine 5′-monophosphate dehydrogenase. Purine metabolism dictates the susceptibility of early morphogenic steps in cytokinesis to NO toxicity.

Crystal structures of the cell-division protein FtsZ from Klebsiella pneumoniae and Escherichia coli

FtsZ, a tubulin-like GTPase, is essential for bacterial cell division. In the presence of GTP, FtsZ polymerizes into filamentous structures, which are key to generating force in cell division. However, the structural basis for the molecular mechanism underlying FtsZ function remains to be elucidated. In this study, crystal structures of the enzymatic domains of FtsZ from Klebsiella pneumoniae (KpFtsZ) and Escherichia coli (EcFtsZ) were determined at 1.75 and 2.50 Å resolution, respectively. Both FtsZs form straight protofilaments in the crystals, and the two structures adopted relaxed (R) conformations. The T3 loop, which is involved in GTP/GDP binding and FtsZ assembly/disassembly, adopted a unique open conformation in KpFtsZ, while the T3 loop of EcFtsZ was partially disordered. The crystal structure of EcFtsZ can explain the results from previous functional analyses using EcFtsZ mutants.

Mutation of G51 in SepF impairs FtsZ assembly promoting ability of SepF and retards the division of Mycobacterium smegmatis cells

The role of FtsZ-associated proteins in the regulation of the assembly dynamics of Mycobacterium smegmatis FtsZ is not clear. In this work, we examined the effect of M. smegmatis SepF on the assembly and stability of M. smegmatis FtsZ polymers. We discovered a single dominant point mutation in SepF (G51D or G51R) that renders the protein inactive. SepF promoted the polymerization of FtsZ, induced the bundling of FtsZ filaments, stabilized FtsZ filaments and reduced the GTPase activity of FtsZ. Surprisingly, both G51D-SepF and G51R-SepF neither stabilized FtsZ filaments nor showed a discernable effect on the GTPase activity of FtsZ. The binding affinity of SepF to FtsZ was found to be stronger than the binding affinity of G51R/D-SepF to FtsZ. Interestingly, the binding affinity of SepF to G51R-SepF was determined to be 45 times stronger than FtsZ. In addition, the interaction of SepF with G51R-SepF was found to be 2.6 times stronger than SepF–SepF interaction. Furthermore, G51R-SepF impaired the ability of SepF to promote the assembly of FtsZ. In addition, the overexpression of G51R-SepF in M. smegmatis mc2 155 cells retarded the proliferation of these cells and increased the average length of the cells. The results indicated that SepF positively regulates the assembly of M. smegmatis FtsZ and the G51 residue has an important role in the functioning of SepF.

Molecular Dynamics Simulation of the E.coli FtsZ

Previous molecular dynamics studies of the FtsZ protein revealed that the protein has high intrinsic flexibility which the crystal structures were unable to reveal. The initial configuration in these studies was based on the available crystal structure data and therefore, the effect of the C-terminal Intrinsically Disordered Region (IDR) of FtsZ could not be observed in these previous studies. Recent investigations have revealed that the C-terminal IDR is crucial for FtsZ assembly in vitro and Z ring formation in vivo. Therefore, in this study, we simulate FtsZ with the IDR.Simulations of the FtsZ monomer in different nucleotide bound forms (without nucleotide, GTP, GDP) were performed. In the conformations of FtsZ monomer with GTP, GTP binds variably with the protein. Such variable interaction with the monomer has not been observed in any previous simulation studies of FtsZ and not observed in crystal structures. The central helix bends towards the C-terminal domain in the GTP bound form, thus making way for polymerization. Nucleotide dependent small shift/rotation of the C-terminal domain was observed in average structures.

Bacterial division FtsZ forms liquid condensates with nucleoid-associated Z-ring inhibitor SlmA

Macromolecular condensation resulting from biologically regulated liquid-liquid phase transitions is emerging as a mechanism to organize the intracellular space in eukaryotic systems, with broad implications in cell physiology and pathology. Here we show that FtsZ, central element of the division ring in most bacteria, forms condensates when in complex with SlmA, the protein preventing septal ring assembly nearby the chromosome in E. coli. The formation of condensates is promoted by crowding and enhanced by sequence-specific binding of SlmA to DNA. These structures are dynamic and FtsZ within them remains active for GTP-triggered fiber formation. Their location is sensitive to compartmentalization and to the presence of a membrane boundary in microfluidics-based cell mimetic systems, likely affecting their reactivity. We propose that reversible condensation may play a role in the modulation of FtsZ assembly and/or location by SlmA and, hence, in the regulation of ring stability, constituting a singular example of a prokaryotic nucleoprotein complex exhibiting this kind of phase transition.