The cell division protein FtsZ is a GTPase structurally related to tubulin and, like tubulin, it assembles in vitro into filaments, sheets and other structures. To study the roles that GTP binding and hydrolysis play in the dynamics of FtsZ polymerization, the nucleotide contents of FtsZ were measured under different polymerizing conditions using a nitrocellulose filter-binding assay, whereas polymerization of the protein was followed in parallel by light scattering. Unpolymerized FtsZ bound 1 mol of GTP mol(-1) protein monomer. At pH 7.5 and in the presence of Mg(2+) and K(+), there was a strong GTPase activity; most of the bound nucleotide was GTP during the first few minutes but, later, the amount of GTP decreased in parallel with depolymerization, whereas the total nucleotide contents remained invariant. These results show that the long FtsZ polymers formed in solution contain mostly GTP. Incorporation of nucleotides into the protein was very fast either when the label was introduced at the onset of the reaction or subsequently during polymerization. Molecular modelling of an FtsZ dimer showed the presence of a cleft between the two subunits maintaining the nucleotide binding site open to the medium. These results show that the FtsZ polymers are highly dynamic structures that quickly exchange the bound nucleotide, and this exchange can occur in all the subunits.
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"FtsZ assembles in the mid-cell into a large structure called the division-ring, which is formed of, among other proteins, polymers of FtsZ . The structure of the monomer–monomer contacts in the FtsZ dimer, including the GTP site in the interface, was predicted (using 3D computer models based on the monomer crystal structure ) to be equivalent to its eukaryotic homologous structure, the tubulin a/b dimer . This hypothesis was later confirmed by crystallography . "
[Show abstract][Hide abstract] ABSTRACT: FtsZ, the prokaryotic ortholog of tubulin, assembles into polymers in the bacterial division ring. The interfaces between monomers contain a GTP molecule, but the relationship between polymerization and GTPase activity is not unequivocally proven. A set of short FtsZ polymers were modelled and the formation of active GTPase structures was monitored using molecular dynamics. Only the interfaces nearest the polymer ends exhibited an adequate geometry for GTP hydrolysis. Simulated conversion of interfaces from close-to-end to internal position and vice versa resulted in their spontaneous rearrangement between active and inactive conformations. This predicted behavior of FtsZ polymer ends was supported by in vitro experiments.
"The fact that the phosphate release starts immediately the GTP is added means that the polymers always contain some GDP. It has been established that the GDP in the polymers exchanges with GTP available in solution . GDP also causes FtsZ protofilament curvature . "
[Show abstract][Hide abstract] ABSTRACT: There is a growing body of evidence that bacterial cell division is an intricate coordinated process of comparable complexity to that seen in eukaryotic cells. The dynamic assembly of Escherichia coli FtsZ in the presence of GTP is fundamental to its activity. FtsZ polymerization is a very attractive target for novel antibiotics given its fundamental and universal function. In this study our aim was to understand further the GTP-dependent FtsZ polymerization mechanism and our main focus is on the pH dependence of its behaviour. A key feature of this work is the use of linear dichroism (LD) to follow the polymerization of FtsZ monomers into polymeric structures. LD is the differential absorption of light polarized parallel and perpendicular to an orientation direction (in this case that provided by shear flow). It thus readily distinguishes between FtsZ polymers and monomers. It also distinguishes FtsZ polymers and less well-defined aggregates, which light scattering methodologies do not. The polymerization of FtsZ over a range of pHs was studied by right-angled light scattering to probe mass of FtsZ structures, LD to probe real-time formation of linear polymeric fibres, a specially developed phosphate release assay to relate guanosine triphosphate (GTP) hydrolysis to polymer formation, and electron microscopy (EM) imaging of reaction products as a function of time and pH. We have found that lowering the pH from neutral to 6.5 does not change the nature of the FtsZ polymers in solution--it simply facilitates the polymerization so the fibres present are longer and more abundant. Conversely, lowering the pH to 6.0 has much the same effect as introducing divalent cations or the FtsZ-associated protein YgfE (a putative ZapA orthologue in E. coli)--it stabilizes associations of protofilaments.
PLoS ONE 06/2011; 6(6):e19369. DOI:10.1371/journal.pone.0019369 · 3.23 Impact Factor
"Either [g-32 P]-GTP or [a-32 P]-GTP was added to the concentrations indicated, and the FtsZ was recovered by filtration or ammonium sulphate precipitation. In the case of the filtration assay, the FtsZ–GTP mixtures (20 ml) were filtered through nitrocellulose disks (pore size 45 mm; Whatman) and washed with 400 ml of wash buffer [20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 5 mM MgSO 4 , 0.005% Triton X-100, 20 mM (NH4) 2 SO 4 ] as described previously (Mingorance et al., 2001). Subsequently, the filters were extracted using 50 ml of ice-cold perchloric acid solution (a 1:2 v/v mixture of 14% perchloric acid, 9 mM EDTA and buffer A). "
[Show abstract][Hide abstract] ABSTRACT: To understand the polymerization dynamics of FtsZ, a bacterial cell division protein similar to tubulin, insight is required into the nature of the nucleotide bound to the polymerized protein. In a previous study, we showed that the FtsZ polymers contain mostly GDP. A recent study challenged this result, suggesting that the polymerized FtsZ is in a GTP-bound state. Here, we show that, when radiolabelled [gamma-32P]-GTP is used to polymerize FtsZ, GTP is hydrolysed instantaneously. The FtsZ polymer contains both GDP and the radiolabelled inorganic phosphate.