The interactions of cell division protein FtsZ with guanine nucleotides

Prokaryotic cell division protein FtsZ, an assembling GTPase, directs the formation of the septosome between daughter cells. FtsZ is an attractive target for the development of new antibiotics. Assembly dynamics of FtsZ is regulated by the binding, hydrolysis, and exchange of GTP. We have determined the energetics of nucleotide binding to model apoFtsZ from Methanococcus jannaschii and studied the kinetics of 2'/3'-O-(N-methylanthraniloyl) (mant)-nucleotide binding and dissociation from FtsZ polymers, employing calorimetric, fluorescence, and stopped-flow methods. FtsZ binds GTP and GDP with K(b) values ranging from 20 to 300 microm(-1) under various conditions. GTP.Mg(2+) and GDP.Mg(2+) bind with slightly reduced affinity. Bound GTP and the coordinated Mg(2+) ion play a minor structural role in FtsZ monomers, but Mg(2+)-assisted GTP hydrolysis triggers polymer disassembly. Mant-GTP binds and dissociates quickly from FtsZ monomers, with approximately 10-fold lower affinity than GTP. Mant-GTP displacement measured by fluorescence anisotropy provides a method to test the binding of any competing molecules to the FtsZ nucleotide site. Mant-GTP is very slowly hydrolyzed and remains exchangeable in FtsZ polymers, but it becomes kinetically stabilized, with a 30-fold slower k(+) and approximately 500-fold slower k(-) than in monomers. The mant-GTP dissociation rate from FtsZ polymers is comparable with the GTP hydrolysis turnover and with the reported subunit turnover in Escherichia coli FtsZ polymers. Although FtsZ polymers can exchange nucleotide, unlike its eukaryotic structural homologue tubulin, GDP dissociation may be slow enough for polymer disassembly to take place first, resulting in FtsZ polymers cycling with GTP hydrolysis similarly to microtubules.     

Energetics of the cooperative assembly of cell division protein FtsZ and the nucleotide hydrolysis switch

FtsZ is the first protein recruited to the bacterial division site, where it forms the cytokinetic Z ring. We have determined the functional energetics of FtsZ assembly, employing FtsZ from the thermophilic Archaea Methanococcus jannaschii bound to GTP, GMPCPP, GDP, or GMPCP, under different solution conditions. FtsZ oligomerizes in a magnesium-insensitive manner. FtsZ cooperatively assembles with magnesium and GTP or GMPCPP into large polymers, following a nucleated condensation polymerization mechanism, under nucleotide hydrolyzing and non-hydrolyzing conditions. The effect of temperature on the critical concentration indicates polymer elongation with an apparent heat capacity change of -800 +/- 100 cal mol-1 K-1 and positive enthalpy and entropy changes, compatible with axial hydrophobic contacts of each FtsZ in the polymer, and predicts optimal polymer stability near 75 degrees C. Assembly entails the binding of one medium affinity magnesium ion and the uptake of one proton per FtsZ. Interestingly, GDP- or GMPCP-liganded FtsZ cooperatively form helically curved polymers, with an elongation only 1-2 kcal mol-1 more unfavorable than the straight polymers formed with nucleotide triphosphate, suggesting a physiological requirement for FtsZ polymerization inhibitors. This GTP hydrolysis switch should provide the basic properties for FtsZ polymer disassembly and its functional dynamics.     

Affinity Labeling of the Guanine Nucleotide Binding Site of Transducin by Pyridoxal 5′-Phosphate

Transducin (T), a guanine nucleotide binding regulatory protein composed of α-, β-, and γ-subunits, serves as an intermediary between rhodopsin and cGMP phosphodiesterase during signaling in the visual process. Pyridoxal 5′-phosphate (PLP), a reagent that has been used to modify enzymes that bind phosphorylated substrates, was probed here as an affinity label for T. PLP inhibited the guanine nucleotide binding activity of T in a concentration dependent manner, and was covalently incorporated into the protein in the presence of [³H]NaBH₄. Approximately 1 mol of ³H was bound per mol of T. GTP and GTP analogs appreciably hindered the incorporation of ³H to T, suggesting that PLP specifically modified the protein active site. Interestingly, PLP modified both the α- and β-subunits of T. Moreover, PLP in the presence of GDP behaved as a GTP analog, since this mixture was capable of dissociating T from T:photoactivated rhodopsin complexes.     

Non-hydrolysable GTP-gamma-S stabilizes the FtsZ polymer in a GDP-bound state

FtsZ, a tubulin homologue, forms a cytokinetic ring at the site of cell division in prokaryotes. The ring is thought to consist of polymers that assemble in a strictly GTP-dependent way. GTP, but not guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S), has been shown to induce polymerization of FtsZ, whereas in vitro Ca2+ is known to inhibit the GTP hydrolysis activity of FtsZ. We have studied FtsZ dynamics at limiting GTP concentrations in the presence of 10 mM Ca2+. GTP and its non-hydrolysable analogue GTP-gamma-S bind FtsZ with similar affinity, whereas the non-hydrolysable analogue guanylyl-imidodiphosphate (GMP-PNP) is a poor substrate. Preformed FtsZ polymers can be stabilized by GTP-gamma-S and are destabilized by GDP. As more than 95% of the nucleotide associated with the FtsZ polymer is in the GDP form, it is concluded that GTP hydrolysis by itself does not trigger FtsZ polymer disassembly. Strikingly, GTP-gamma-S exchanges only a small portion of the FtsZ polymer-bound GDP. These data suggest that FtsZ polymers are stabilized by a small fraction of GTP-containing FtsZ subunits. These subunits may be located either throughout the polymer or at the polymer ends, forming a GTP cap similar to tubulin.     

Understanding Nucleotide-Regulated FtsZ Filament Dynamics and the Monomer Assembly Switch with Large-Scale Atomistic Simulations

Bacterial cytoskeletal protein FtsZ assembles in a head-to-tail manner, forming dynamic filaments that are essential for cell division. Here, we study their dynamics using unbiased atomistic molecular simulations from representative filament crystal structures. In agreement with experimental data, we find different filament curvatures that are supported by a nucleotide-regulated hinge motion between consecutive FtsZ monomers. Whereas GTP-FtsZ filaments bend and twist in a preferred orientation, thereby burying the nucleotide, the differently curved GDP-FtsZ filaments exhibit a heterogeneous distribution of open and closed interfaces between monomers. We identify a coordinated Mg²⁺ ion as the key structural element in closing the nucleotide site and stabilizing GTP filaments, whereas the loss of the contacts with loop T7 from the next monomer in GDP filaments leads to open interfaces that are more prone to depolymerization. We monitored the FtsZ monomer assembly switch, which involves opening/closing of the cleft between the C-terminal domain and the H7 helix, and observed the relaxation of isolated and filament minus-end monomers into the closed-cleft inactive conformation. This result validates the proposed switch between the low-affinity monomeric closed-cleft conformation and the active open-cleft FtsZ conformation within filaments. Finally, we observed how the antibiotic PC190723 suppresses the disassembly switch and allosterically induces closure of the intermonomer interfaces, thus stabilizing the filament. Our studies provide detailed structural and dynamic insights into modulation of both the intrinsic curvature of the FtsZ filaments and the molecular switch coupled to the high-affinity end-wise association of FtsZ monomers.     

Understanding Nucleotide-Regulated FtsZ Filament Dynamics and the Monomer Assembly Switch with Large-Scale Atomistic Simulations

Bacterial cytoskeletal protein FtsZ assembles in a head-to-tail manner, forming dynamic filaments that are essential for cell division. Here, we study their dynamics using unbiased atomistic molecular simulations from representative filament crystal structures. In agreement with experimental data, we find different filament curvatures that are supported by a nucleotide-regulated hinge motion between consecutive FtsZ monomers. Whereas GTP-FtsZ filaments bend and twist in a preferred orientation, thereby burying the nucleotide, the differently curved GDP-FtsZ filaments exhibit a heterogeneous distribution of open and closed interfaces between monomers. We identify a coordinated Mg²⁺ ion as the key structural element in closing the nucleotide site and stabilizing GTP filaments, whereas the loss of the contacts with loop T7 from the next monomer in GDP filaments leads to open interfaces that are more prone to depolymerization. We monitored the FtsZ monomer assembly switch, which involves opening/closing of the cleft between the C-terminal domain and the H7 helix, and observed the relaxation of isolated and filament minus-end monomers into the closed-cleft inactive conformation. This result validates the proposed switch between the low-affinity monomeric closed-cleft conformation and the active open-cleft FtsZ conformation within filaments. Finally, we observed how the antibiotic PC190723 suppresses the disassembly switch and allosterically induces closure of the intermonomer interfaces, thus stabilizing the filament. Our studies provide detailed structural and dynamic insights into modulation of both the intrinsic curvature of the FtsZ filaments and the molecular switch coupled to the high-affinity end-wise association of FtsZ monomers.     

Calculations of binding affinity between C8-substituted GTP analogs and the bacterial cell-division protein FtsZ

The FtsZ protein is a self-polymerizing GTPase that plays a central role in bacterial cell division. Several C8-substituted GTP analogs are known to inhibit the polymerization of FtsZ by competing for the same binding site as its endogenous activating ligand GTP. Free energy calculations of the relative binding affinities to FtsZ for a set of five C8-substituted GTP analogs were performed. The calculated values agree well with the available experimental data, and the main contribution to the free energy differences is determined to be the conformational restriction of the ligands. The dihedral angle distributions around the glycosidic bond of these compounds in water are known to vary considerably depending on the physicochemical properties of the substituent at C8. However, within the FtsZ protein, this substitution has a negligible influence on the dihedral angle distributions, which fall within the narrow range of −140° to −90° for all investigated compounds. The corresponding ensemble average of the coupling constants ³ J(C4,H1′) is calculated to be 2.95 ± 0.1 Hz. The contribution of the conformational selection of the GTP analogs upon binding was quantified from the corresponding populations. The obtained restraining free energy values follow the same trend as the relative binding affinities to FtsZ, indicating their dominant contribution.     

Characterization of Caulobacter crescentus FtsZ protein using dynamic light scattering

The self-assembly of the tubulin homologue FtsZ at the mid-cell is a critical step in bacterial cell division. We introduce dynamic light scattering (DLS) spectroscopy as a new method to study the polymerization kinetics of FtsZ in solution. Analysis of the DLS data indicates that the FtsZ polymers are remarkably monodisperse in length, independent of the concentrations of GTP, GDP, and FtsZ monomers. Measurements of the diffusion coefficient of the polymers demonstrate that their length is remarkably stable until the free GTP is consumed. We estimated the mean size of the FtsZ polymers within this interval of stable length to be between 9 and 18 monomers. The rates of FtsZ polymerization and depolymerization are likely influenced by the concentration of GDP, as the repeated addition of GTP to FtsZ increased the rate of polymerization and slowed down depolymerization. Increasing the FtsZ concentration did not change the size of FtsZ polymers; however, it increased the rate of the depolymerization reaction by depleting free GTP. Using transmission electron microscopy we observed that FtsZ forms linear polymers in solutions which rapidly convert to large bundles upon contact with surfaces at time scales as short as several seconds. Finally, the best studied small molecule that binds to FtsZ, PC190723, had no stabilizing effect on Caulobacter crescentus FtsZ filaments in vitro, which complements previous studies with Escherichia coli FtsZ and confirms that this class of small molecules binds Gram-negative FtsZ weakly.     

The transitory complex between photoexcited rhodopsin and transducin. Reciprocal interaction between the retinal site in rhodopsin and the nucleotide site in transducin

In the first step of the visual transduction cascade a photoexcited rhodopsin molecule, R*ret, binds to a GDP-carrying transducin molecule, TGDP. The R*-T interaction causes the opening of the nucleotide site in T and catalyzes the GDP/GTP exchange by allowing the release of the GDP. We have studied the influences on this R*-T transitory complex of the occupancies of the nucleotide site in T and the retinal site in rhodopsin. After elimination of the GDP released from the bound transducin, the complex, named R*ret-te (ret for retinal present, e for nucleotide site empty) remains stabilized almost indefinitely in a medium whose ionic composition is close to physiological. In this complex the bound Te retains a lasting ability to interact with GDP or GTP, and R*ret remains spectroscopically in the meta-II state, by contrast with free R*ret which decays to opsin and free retinal. Hence the R*-T interaction which opens the nucleotide site in T conversely blocks the retinal site in R*ret. Upon prolonged incubation in a low-ionic-strength medium the R*ret-Tc complex dissociates partially, but the liberated Te is then unable to rebind GDP or GTP, even in the presence of R*ret, it is probably denaturated. Upon treatment of the R*ret-Te complex by a high concentration of hydroxylamine, the retinal can be removed from the rhodopsin. The Re-Te complex remains stable and the complexed transducin keeps its capacity to bind GTP. TGTP then dissociates from Re. The liberated Re loses its capacity to interact with a new transducin. These data are integrated into a discussion of the development of the cascade. We stress that affinities, i.e. dissociation equilibrium constants, are insufficient to describe the flow of reactions triggered by one R*ret molecule. It depends on a few critical rapid binding and dissociation processes, and is practically insensitive to other slow ones, hence to the values of affinities that express only the ratio of kinetics constants. The effect of the R*-T interaction on the retinal site in rhodopsin is analogous to the effect of the binding of a G-protein on the apparent affinity of a receptor for its agonist.     

Nucleotide recognition by the initiation factor aIF5B: Free energy simulations of a neoclassical GTPase

The GTPase aIF5B is a universally conserved initiation factor that assists ribosome assembly. Crystal structures of its nucleotide complexes, X‐ray(GTP) and X‐ray(GDP), are similar in the nucleotide vicinity, but differ in the orientation of a distant domain IV. This has led to two, contradictory, mechanistic models. One postulates that X‐ray(GTP) and X‐ray(GDP) are, respectively, the active, “ON” and the inactive, “OFF” states; the other postulates that both structures are OFF, whereas the ON state is still uncharacterized. We study GTP/GDP binding using molecular dynamics and a continuum electrostatic free energy method. We predict that X‐ray(GTP) has a ≈ 3 kcal/mol preference to bind GDP, apparently contradicting its assignment as ON. However, the preference arises mainly from a single, nearby residue from the switch 2 motif: Glu81, which becomes protonated upon GTP binding, with a free energy cost of about 4 kcal/mol. We then propose a different model, where Glu81 protonation/deprotonation defines the ON/OFF states. With this model, the X‐ray(GTP):GTP complex, with its protonated Glu81, is ON, whereas X‐ray(GTP):GDP is OFF. The model postulates that distant conformational changes such as domain IV rotation are “uncoupled” from GTP/GDP exchange and do not affect the relative GTP/GDP binding affinities. We analyze the model using a general thermodynamic framework for GTPases. It yields rather precise predictions for the nucleotide specificities of each state, and the state specificities of each nucleotide, which are roughly comparable to the homologues IF2 and aIF2, despite the lack of any conformational switching in the model. © 2012 Wiley Periodicals, Inc.     

Rapid irreversible G protein alpha subunit misfolding due to intramolecular kinetic bottleneck that precedes Mg2+ "lock" after GTP/GDP exchange

Stoichiometric exchange of GTP for GDP on heterotrimeric G protein alpha (Galpha) subunits is essential to most hormone and neurotransmitter initiated signal transduction. Galphas are stably activated in a Mg2+ complex with GTPgammaS, a nonhydrolyzable GTP analogue that is reported to bind Galpha, with very high affinity. Yet, it is common to find that substantial amounts (30-90%) of purified G proteins cannot be activated. Inactivatable G protein has heretofore been thought to have become "denatured" during formation of the obligatory nucleotide-free or empty (MT) Galpha-state that is intermediary to GDP/GTP exchange at a single binding site. We find Galpha native secondary and tertiary structure to persist during formation of the irreversibly inactivatable state of transducin. MT Galpha is therefore irreversibly misfolded rather than denatured. Inactivation by misfolding is found to compete kinetically with protective but weak preequilibrium nucleotide binding at micromolar ambient GTPgammaS concentrations. Because of the weak preequilibrium, quantitative protection against Galpha aggregation is only achieved at free nucleotide concentrations 10-100 times higher than those commonly employed in G protein radio-nucleotide binding studies. Initial GTP protection is also poor because of the extreme slowness of an intramolecular Galpha refolding step (isomerization) necessary for GTP sequestration after its weak preequilibrium binding. Of the two slowly interconverting Galpha x GTP isomers described here, only the second can bind Mg2+, "locking" GTP in place with a large net rise in GTP binding affinity. A companion Galpha x GDP isomerization reaction is identified as the cause of the very slow spontaneous GDP dissociation that characterizes G protein nucleotide exchange and low spontaneous background activity in the absence of GPCR activation. Galpha x GDP and Galpha x GTP isomerization reactions are proposed as the dual target for GPCR catalysis of nucleotide exchange.     

GTP/GDP binding stabilizes bacterial cell division protein FtsZ against degradation by FtsH protease in vitro

Factors contributing to the stability of bacterial cell division protein FtsZ remain unknown. In order to identify FtsZ-stabilizing factor(s), we exploited FtsH protease-based in vitro FtsZ degradation assay system. Whole cell lysate from an ftsH-null strain of Escherichia coli inhibited degradation of FtsZ by FtsH in vitro. However, activated charcoal-treated lysate did not inhibit degradation. The loss of ability of the activated charcoal-treated lysate to inhibit degradation of FtsZ was restored when it was replenished with GTP, but not when replenished with other NTPs or dNTPs. The lysate did not protect either FtsZ deletion mutants, which do not bind GTP, or FtsH substrates, sigma(32) and cI-108 proteins, against FtsH. GDP and GTPgammaS also stabilized FtsZ against FtsH. Neither GTP nor GDP inhibited proteolytic activity of FtsH per se. These observations demonstrate that binding of GTP/GDP ligands is responsible for the proteolytic stability of FtsZ against FtsH.     

Crystal structure of transglutaminase 3 in complex with GMP: structural basis for nucleotide specificity

Epidermal-type Transglutaminase 3 (TGase 3) is a Ca(2+)-dependent enzyme involved in the cross-linking of structural proteins required in the assembly of the cell envelope. We have recently shown that calcium-activated TGase 3, like TGase 2, can bind, hydrolyze, and is inhibited by GTP despite lacking structural homology with other GTP-binding proteins. Here we report the crystal structure determined at 2.0 A resolution of TGase 3 in complex with GMP to elucidate the structural features required for nucleotide recognition. Binding affinities for various nucleotides were found by fluorescence displacement to be as follows: guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) (0.4 microm), GTP (0.6 microm), GDP (1.0 microm), GMP (0.4 microm), and ATP (28.0 microm). Furthermore, we found that GMP binds as a reversible, noncompetitive inhibitor of TGase 3 transamidation activity, similar to GTPgammaS and GDP. A genetic algorithm similarity program (GASP) approach (virtual ligand screening) identified three compounds from the Lead Quest trade mark data base (Tripos Inc.) based on superimposition of GTPgammaS, GDP, and GMP guanine nucleotides from our crystal structures to generate the minimum align flexible fragment. These three were nucleotide analogs without a phosphate group containing the minimal binding motif for TGase 3 that includes a nucleoside recognition groove. Binding affinities were measured as follows: TP349915 (K(d) = 4.1 microm), TP395289 (K(d) = 38.5 microm), TP394305 (K(d) = 1.0 mm). Remarkably, these compounds do not inhibit but instead activate TGase 3 transamidation by about 10-fold. These results suggest that the nucleotide binding pocket in TGase 3 may be exploited to either enhance or inhibit the enzymatic activity as required for different therapeutic approaches.     

Polymerization of nucleotide-free, GDP- and GTP-bound cell division protein FtsZ: GDP makes the difference

Stable, more than 98% nucleotide-free apo-FtsZ was prepared from purified Methanococcus jannaschhi FtsZ. This facilitates the study of the functional mechanisms of this FtsZ, an assembling GTPase, which shares a common fold with eukaryotic tubulin. Apo-FtsZ underwent cooperative magnesium-induced polymerization with a similar critical concentration and morphology related to that of reconstituted GTP-bound FtsZ, suggesting that the binding of GTP contributes insignificantly to the stability of the FtsZ polymers. On the other hand, reconstituted GDP-FtsZ polymerized with a larger critical concentration than GTP-FtsZ, indicating that GDP binding destabilizes FtsZ polymers. Upon GTP hydrolysis by FtsZ polymers, in the absence of a continued GTP supply and under macromolecular crowding conditions enhancing FtsZ polymerization, the straight GTP polymers disappeared and were replaced by characteristic helically curved GDP-bound polymers. These results suggest that the roles of GTP binding and hydrolysis by this archaeal FtsZ are simply to facilitate disassembly. In a physiological situation in GTP excess, GDP-bound FtsZ subunits could again bind GTP, or trigger disassembly, or be recognized by FtsZ filament depolymerizing proteins, allowing the Z-ring dynamics during prokaryotic cell division.     

Independence between GTPase active sites in the Escherichia coli cell division protein FtsZ

We have analyzed the substrate kinetics of the GTPase activity of FtsZ and the effects of two different GTPase inhibitors, GDP and the slowly hydrolyzable GTP analogue GMPCPP. In the absence of inhibitors the GTPase activity follows simple Michaelis-Menten kinetics, and both GDP and GMPCPP inhibited the activity in a competitive manner. These results indicate that the GTPase active sites in FtsZ filaments are independent of each other, a feature relevant to elucidate the role of GTP hydrolysis in FtsZ function and cell division.     

Assembly of archaeal cell division protein FtsZ and a GTPase-inactive mutant into double-stranded filaments

We have studied the assembly and GTPase of purified FtsZ from the hyperthermophilic archaeon Methanococcus jannaschii, a structural homolog of eukaryotic tubulin, employing wild-type FtsZ, FtsZ-His6 (histidine-tagged FtsZ), and the new mutants FtsZ-W319Y and FtsZ-W319Y-His6, with light scattering, nucleotide analyses, electron microscopy, and image processing methods. This has revealed novel properties of FtsZ. The GTPase of archaeal FtsZ polymers is suppressed in Na+-containing buffer, generating stabilized structures that require GDP addition for disassembly. FtsZ assembly is polymorphic. Archaeal FtsZ(wt) assembles into associated and isolated filaments made of two parallel protofilaments with a 43 A longitudinal spacing between monomers, and this structure is also observed in bacterial FtsZ from Escherichia coli. The His6 extension facilitates the artificial formation of helical tubes and sheets. FtsZ-W319Y-His6 is an inactivated GTPase whose assembly remains regulated by GTP and Mg2+. It forms two-dimensional crystals made of symmetrical pairs of tubulin-like protofilaments, which associate in an antiparallel array (similarly to the known Ca2+-induced sheets of FtsZ-His6). In contrast to the lateral interactions of microtubule protofilaments, we propose that the primary assembly product of FtsZ is the double-stranded filament, one or several of which might form the dynamic Z ring during prokaryotic cell division.     

Stereochemistry of the guanyl nucleotide binding site of transducin probed by phosphorothioate analogues of GTP and GDP

The stereochemistry of the guanyl nucleotide binding site of transducin from bovine retinal rod outer segments was probed with phosphorothioate analogues of GTP and GDP. Transducin has markedly different affinities for the five thio analogues of GTP, as measured by their effectiveness in inhibiting GTPase activity, competing with GTP for entry into transducin, and displacing GDP bound to transducin. The order of binding affinities is GTP gamma S = (Sp)-GTP alpha S greater than (Rp)-GTP alpha S greater than (Sp)-GTP beta S much greater than (Rp)-GTP beta S. The affinity of transducin for GTP gamma S is greater than 10(4) higher than that for (Rp)-GTP beta S. These five analogues have the same relative potencies in eliciting the release of transducin from the membrane and in activating the phosphodiesterase. Transducin hydrolyzes (Sp)-GTP alpha S with a l/e time of 55 s, compared with 28 s for GTP. In contrast, (Rp)-GTP alpha S, like GTP gamma S, is not hydrolyzed on the time scale of several hours. The order of effectiveness of thio analogues of GDP in displacing bound GDP is (Sp)-GDP alpha S greater than GDP greater than (Rp)-GDP alpha S greater than GDP beta S. The affinity of transducin for (Sp)-GDP alpha S is about 10-fold higher than that for GDP beta S. Mg2+ is required for the binding of GTP and GDP to transducin. Cd2+ does not lead to a reversal of stereospecificity at either the alpha- or beta-phosphorus atom of GTP. These results lead to the following conclusions: The pro-R oxygen atom at the alpha-phosphorus of GTP does not bind Mg2+ but instead interacts with the protein. The pro-S oxygen at the alpha-phosphorus does not appear to be involved in a critical interaction with transducin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substitution of a conserved aspartate allows cation-induced polymerization of FtsZ

The prokaryotic tubulin homologue FtsZ polymerizes in vitro in a nucleotide dependent fashion. Here we report that replacement of the strictly conserved Asp212 residue of Escherichia coli FtsZ by a Cys or Asn, but not by a Glu residue results in FtsZ that polymerizes with divalent cations in the absence of added GTP. FtsZ D212C and D212N mutants co-purify with GTP as bound nucleotide, providing an explanation for the unusual phenotype. We conclude that D212 plays a critical role in the coordination of a metal ion and the nucleotide at the interface of two FtsZ monomers.     

R-loop-dependent hypernegative supercoiling in Escherichia coli topA mutants preferentially occurs at low temperatures and correlates with growth inhibition

hGBP1 is a GTPase with antiviral activity encoded by an interferon- activated human gene. Specific binding of hGBP1 to guanine nucleotides has been established although only two classical GTP-binding motifs were found in its primary sequence. The unique position of hGBP1 amongst known GTPases is further demonstrated by the hydrolysis of GTP to GDP and GMP. Although subsequent cleavage of orthophosphates rather than pyrophosphate was demonstrated, GDP coming from bulk solution cannot serve as a substrate. The relation of guanine nucleotide binding and hydrolysis to the antiviral function of hGBP1 is unknown. Here we show similar binding affinities for all three guanine nucleotides and the ability of both products, GDP and GMP, to compete with GTP binding. Fluorimetry and isothermal titration calorimetry were applied to prove that only one nucleotide binding site is present in hGBP1. Furthermore, we identified the third canonical GTP-binding motif and verified its role in nucleotide recognition by mutational analysis. The high guanine nucleotide dissociation rates measured by stopped-flow kinetics are responsible for the weak affinities to hGBP1 when compared to other GTPases like Ras or Galpha. By means of fluorescence and NMR spectroscopy it is demonstrated that aluminium fluoride forms a complex with hGBP1 only in the GDP state, presumably mimicking the transition state of GTP hydrolysis. Tentatively, the involvement of a GAP domain in hGBP1 in GTP hydrolysis is suggested. These results will serve as a basis for the determination of the differential biological functions of the three nucleotide states and for the elucidation of the unique mechanism of nucleotide hydrolysis catalysed by hGBP1.     

Activation of cell division protein FtsZ. Control of switch loop T3 conformation by the nucleotide gamma-phosphate

The effect of bound nucleotide on the conformation of cell division protein FtsZ from Methanococcus jannaschii has been investigated using molecular dynamics and site-directed mutagenesis. The molecular dynamics indicate that the gamma-phosphate of GTP induces a conformational perturbation in loop T3 (Gly88-Gly99 segment), in a position structurally equivalent to switch II of Ha-ras-p21. In the simulated GTP-bound state, loop T3 is pulled by the gamma-phosphate into a more compact conformation than with GDP, related to that observed in the homologous proteins alpha- and beta-tubulin. The existence of a nucleotide-induced structural change in loop T3 has been confirmed by mutating Thr92 into Trp (T92W-W319Y FtsZ). This tryptophan (12 A away from gamma-phosphate) shows large differences in fluorescence emission, depending on which nucleotide is bound to FtsZ monomers. Loop T3 is located at a side of the contact interface between two FtsZ monomers in the current model of FtsZ filament. Such a structural change may bend the GDP filament upon hydrolysis by pushing against helix H8 of next monomer, thus, generating force on the membrane during cell division. A related curvature mechanism may operate in tubulin activation.