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Nucleotide-dependent conformations of FtsZ dimers and force generation observed through molecular dynamics simulations.

ABSTRACT: The bacterial cytoskeletal protein FtsZ is a GTPase that is thought to provide mechanical constriction force via an unidentified mechanism. Purified FtsZ polymerizes into filaments with varying structures in vitro: while GTP-bound FtsZ assembles into straight or gently curved filaments, GDP-bound FtsZ forms highly curved filaments, prompting the hypothesis that a difference in the inherent curvature of FtsZ filaments provides mechanical force. However, no nucleotide-dependent structural transition of FtsZ monomers has been observed to support this force generation model. Here, we present a series of all-atom molecular dynamics simulations probing the effects of nucleotide binding on the structure of an FtsZ dimer. We found that the FtsZ-dimer structure is dependent on nucleotide-binding state. While a GTP-bound FtsZ dimer retained a firm monomer-monomer contact, a GDP-bound FtsZ dimer lost some of the monomer-monomer association, leading to a "hinge-opening" event that resulted in a more bent dimer, while leaving each monomer structure largely unaffected. We constructed models of FtsZ filaments and found that a GDP-FtsZ filament is much more curved than a GTP-FtsZ filament, with the degree of curvature matching prior experimental data. FtsZ dynamics were used to estimate the amount of force an FtsZ filament could exert when hydrolysis occurs (20-30 pN per monomer). This magnitude of force is sufficient to direct inward cell-wall growth during division, and to produce the observed degree of membrane pinching in liposomes. Taken together, our data provide molecular-scale insight on the origin of FtsZ-based constriction force, and the mechanism underlying prokaryotic cell division.

PROVIDER: S-EPMC3386107 | BioStudies |

REPOSITORIES: biostudies

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Project description:To divide, bacteria must constrict their membranes against significant force from turgor pressure. A tubulin homolog, FtsZ, is thought to drive constriction, but how FtsZ filaments might generate constrictive force in the absence of motor proteins is not well understood. There are two predominant models in the field. In one, FtsZ filaments overlap to form complete rings around the circumference of the cell, and attractive forces cause filaments to slide past each other to maximize lateral contact. In the other, filaments exert force on the membrane by a GTP-hydrolysis-induced switch in conformation from straight to bent. Here, we developed software, ZCONSTRICT, for quantitative three-dimensional (3D) simulations of Gram-negative bacterial cell division to test these two models and identify critical conditions required for them to work. We find that the avidity of any kind of lateral interactions quickly halts the sliding of filaments, so a mechanism such as depolymerization or treadmilling is required to sustain constriction by filament sliding. For filament bending, we find that a mechanism such as the presence of a rigid linker is required to constrain bending to within the division plane and maintain the distance observed <i>in vivo</i> between the filaments and the membrane. Of these two models, only the filament bending model is consistent with our lab's recent observation of constriction associated with a single, short FtsZ filament.<b>IMPORTANCE</b> FtsZ is thought to generate constrictive force to divide the cell, possibly via one of two predominant models in the field. In one, FtsZ filaments overlap to form complete rings which constrict as filaments slide past each other to maximize lateral contact. In the other, filaments exert force on the membrane by switching conformation from straight to bent. Here, we developed software, ZCONSTRICT, for three-dimensional (3D) simulations to test these two models. We find that a mechanism such as depolymerization or treadmilling are required to sustain constriction by filament sliding. For filament bending, we find that a mechanism that constrains bending to within the division plane is required to maintain the distance observed <i>in vivo</i> between the filaments and the membrane.

Project description:<h4>Introduction</h4>Nucleoside diphosphate kinase (NDK), conserved across bacteria to humans, synthesises NTP from NDP and ATP. The eukaryotic homologue, the NDPK, uses ATP to phosphorylate the tubulin-bound GDP to GTP for tubulin polymerisation. The bacterial cytokinetic protein FtsZ, which is the tubulin homologue, also uses GTP for polymerisation. Therefore, we examined whether NDK can interact with FtsZ to convert FtsZ-bound GDP and/or free GDP to GTP to trigger FtsZ polymerisation.<h4>Methods</h4>Recombinant and native NDK and FtsZ proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis were used as the experimental samples. FtsZ polymersation was monitored using 90° light scattering and FtsZ polymer pelleting assays. The ?32P-GTP synthesised by NDK from GDP and ?32P-ATP was detected using thin layer chromatography and quantitated using phosphorimager. The FtsZ bound 32P-GTP was quantitated using phosphorimager, after UV-crosslinking, followed by SDS-PAGE. The NDK-FtsZ interaction was determined using Ni2+-NTA-pulldown assay and co-immunoprecipitation of the recombinant and native proteins in vitro and ex vivo, respectively.<h4>Results</h4>NDK triggered instantaneous polymerisation of GDP-precharged recombinant FtsZ in the presence of ATP, similar to the polymerisation of recombinant FtsZ (not GDP-precharged) upon the direct addition of GTP. Similarly, NDK triggered polymerisation of recombinant FtsZ (not GDP-precharged) in the presence of free GDP and ATP as well. Mutant NDK, partially deficient in GTP synthesis from ATP and GDP, triggered low level of polymerisation of MsFtsZ, but not of MtFtsZ. As characteristic of NDK's NTP substrate non-specificity, it used CTP, TTP, and UTP also to convert GDP to GTP, to trigger FtsZ polymerisation. The NDK of one mycobacterial species could trigger the polymerisation of the FtsZ of another mycobacterial species. Both the recombinant and the native NDK and FtsZ showed interaction with each other in vitro and ex vivo, alluding to the possibility of direct phosphorylation of FtsZ-bound GDP by NDK.<h4>Conclusion</h4>Irrespective of the bacterial species, NDK interacts with FtsZ in vitro and ex vivo and, through the synthesis of GTP from FtsZ-bound GDP and/or free GDP, and ATP (CTP/TTP/UTP), triggers FtsZ polymerisation. The possible biological context of this novel activity of NDK is presented.

Project description:Microtubules are long polymers of alphabeta-tubulin heterodimers. They undergo a process known as dynamic instability, in which the ends of a microtubule switch stochastically between phases of slow growth and rapid shrinkage. The molecular mechanisms inducing the depolymerization of microtubules were attributed to the hydrolysis of the guanosine triphosphate (GTP) nucleotide bound to the beta-tubulin. The hydrolysis of GTP is thought to cause microtubule instability by promoting outward curving of the protofilaments constituting the microtubule lattice. The bending of protofilaments is associated with the structural transformation of a tubulin dimer from straight to curved conformations. However, the nature of intrinsic bending of the dimer remains elusive. This study uses molecular dynamics (MD) simulations and coarse-grained analysis to reveal the intrinsic bending, as well as the local structural rearrangements, of the unassembled tubulin dimer as the dimer relaxes from its lattice-constrained, straight conformation of a zinc-induced tubulin sheet. The effect of the nucleotide state on dimer-bending is investigated by the introduction of gamma-phosphate into the beta-tubulin to form GTP-bound tubulin. In agreement with recent experimental studies that proposed nucleotide-independent curved conformations, both guanosine diphosphate (GDP)-bound and GTP-bound tubulin dimers were found to have curved conformations, but with a tendency toward smaller bending in the GTP-tubulin than in the GDP-tubulin. The perturbation induced through the introduction of gamma-phosphate is posited to play a role in straightening the intradimer bending. The local structural rearrangements of GDP-tubulin because of the bending mode of motion of the dimer reveal that one of the three functional domains, the intermediate domain, exhibits significantly lower bending deformation compared with the others, signifying a dynamic connection to the functionally defined domains.

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Nucleotide-dependent conformations of FtsZ dimers and force generation observed through molecular dynamics simulations.

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