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 Å 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.

Bacterial cells place at their division site a Z-ring that assembles from FtsZ at the cytosolic side of the plasma membrane and recruits the other protein components of the septation machinery. The positioning of the Z-ring is in turn determined by the self-organizing Min system (1)(2)(3). Archaeal FtsZ from Methanococcus jannaschii and eukaryotic tubulin share the same structural framework and constitute a distinct family of protofilament-forming GTPases (4,5). The structures of bacterial FtsZs are probably similar to the archaeal protein, based on FtsZ sequence conservation. The Escherichia coli Z-ring is very dynamic, with turnover in seconds, similar to or faster than a typical mitotic spindle (6). Although the position of the Z ring was visualized by immunoelectron microscopy (7), its structure is unknown. A number of studies have shown that purified bacterial FtsZ can assemble with GTP and divalent cations forming polymers that hydrolyze the nucleotide ␤-␥ phosphate bond and disassemble upon GTP consumption (8 -13), although the mechanism linking GTP hydrolysis and exchange to the polymer dynamics (14) remains controversial (15)(16)(17). FtsZ polymers have the characteristic tubulin ϳ40 Å axial spacing between consecutive monomers (18). At pH 6 and in the presence of high Ca 2ϩ and GTP concentrations, a small proportion of archaeal FtsZ from M. jannaschii formed crystalline sheets made of symmetric protofilament pairs associated in an antiparallel arrangement, whereas most of the protein precipitated into aggregates of helical cable-like tubes. Manual fitting of the structure of GDP-bound FtsZ from M. jannaschii (19) into a threedimensional electron microscopy map of the sheet polymers has led to the current tubulin-like FtsZ protofilament model (5). Purified bacterial FtsZ without macromolecular additives or Ca 2ϩ assembles into polymers apparently made of two or several protofilaments (9,12,16,20,21), although single-stranded segments can also be observed in negative stain, and employing scanning transmission electron microscopy, at low FtsZ concentrations single-stranded polymers have been reported (15).
FtsZ folding, activation, and assembly have important differences with tubulins. Purified unfolded FtsZ polypeptide chains are capable of spontaneous refolding into their functional states (21) in contrast to tubulin monomers, whose folding process includes binding and release from cytoplasmic chaperonin CCT (22). We have hypothesized that the extensive tubulin loop insertions in the FtsZ/tubulin common core and its folding complexity coevolved with the lateral association interfaces responsible for the association of parallel protofilaments into microtubules, suggesting that tubulin and FtsZ share a similar axial interaction between monomers, but have different interactions between protofilaments (21). Employing molecular dynamics it has been predicted that GTP binding controls the conformation of switch loop T3, which activates FtsZ for assembly possibly in a manner similar to tubulin (23). In this work we have investigated the assembly of archaeal FtsZ from M. jannaschii. We started by defining the conditions for biochemically regulated assembly of this FtsZ, and show that the GTPase of FtsZ is activated by polymerization, and that GDP induces disassembly. Subsequently, results not found before with other FtsZ proteins have provided new insight into FtsZ assembly. Archaeal FtsZ assembles without hydrolyzing GTP in sodiumcontaining buffer. The mutation W319Y also suppresses the GTPase activity of the archaeal FtsZ, generating stabilized polymers and two-dimensional crystals. Archaeal FtsZ assemblies are made of symmetrical pairs of tubulin-like protofilaments, which associate in a manner different than that of tubulin. We propose that these double-stranded polymers constitute the primary assembly product of archaeal FtsZ and show that they are also formed in bacterial FtsZ from E. coli.

EXPERIMENTAL PROCEDURES
FtsZ, FtsZ-His 6 , FtsZ-W319Y, and FtsZ-W319Y-His 6 Proteins-FtsZ-His 6 from M. jannaschii, which is wild-type FtsZ (FTSZ1) with a Cterminal Gly-Ser-His 6 extension, and its mutant FtsZ-W319Y-His 6 , were overproduced in E. coli BL21(DE3)pLys transformed with the plasmid pHis17-mjFtsZ-H 6 (19,24) or pAVK1(W319Y) (23), respectively, and purified by HiTrap Ni 2ϩ -chelating affinity and size-exclusion chromatography as described (21,23). The FtsZ-W319Y-His 6 mutant had been originally constructed as an intermediate step to the double mutant FtsZ-W319Y-T92W-His 6 , whose single tryptophan fluorescence is sensitive to GDP or GTP binding (23). Plasmids coding for FtsZ and FtsZ-W319Y, both without the Gly-Ser-His 6 extension, were respectively constructed using the plasmids pHis17-mjFtsZ-H 6 or pAVK1(W319Y) as templates, by inverse PCR with the Expand High Fidelity PCR system (Roche Applied Science) in a GeneAmp PCR 2400 (PerkinElmer Life Sciences), employing the synthetic primers TA-AAAGCTTCACCACCACCACCACCA and AATTTTTGGAATTCCTGT-GAGTTC, which results in the loss of the sequence coding for the Gly-Ser-His 6 extension and the BamH1 restriction site. The PCR parameters were 1 cycle of 5 s at 95°C, 15 min at 50°C, 4 min at 68°C, followed by 14 cycles of 15 s at 95°C, 30 s at 50°C and 4 min at 68°C, terminating with 20 min at 68°C. The PCR products were purified with the Concert Rapid PCR purification system (Invitrogen), phosphorylated with phage T4 kinase (Promega), polished with DNA polymerase Pwo (Roche Applied Science), ligated with T4 DNA ligase (Promega), and the parental plasmid digested with DpnI (Promega). E. coli DH5␣ cells were transformed with the ligated products, plasmid DNA was purified from individual transformants and employed to transform E. coli BL21(DE3) pLys for overexpression. The mutant plasmids were identified by the loss of the BamH1 restriction site of the parental DNA and by sequencing their ftsZ gene, employing the sequencing primers mf3 (ACCATATCTGAATCTTG), mj1 (CGGCATATTTGGAAC), p319y (ATATATAATTGTAGCATTTGGGTCTAATCTTG) and mj3 (GAA-CAATGGAGGCTTAGCG) with an Applied Biosystems PRISM 3700 DNA analyzer. The absence of the histidine tags in FtsZ and FtsZ-W319Y was confirmed by immunoblotting with anti-His antibody (Amersham Biosciences).
FtsZ and FtsZ-W319Y were overexpressed as described above for the His tag-containing proteins, but they were precipitated from the high speed supernatants of the cell lysates with 33% ammonium sulfate. The precipitate was resuspended in 50 mM Tris-HCl, 1 mM EDTA, 150 mM KCl, pH 8.0, clarified, and chromatographed in a HiTrap Q-HP anion exchange column (5 ml, Amersham Biosciences) with a gradient of 0.15-1.0 M KCl in the same buffer. FtsZ, which eluted at ϳ0.45 M KCl together with a ϳ98 kDa protein contaminant, was concentrated by ultrafiltration with Centriprep 30K (Amicon), and it was further purified by hydrophobic chromatography in a HiTrap Phenyl Sepharose HP column (1 ml, Amersham Biosciences) with a 0.8 -0 M ammonium sulfate gradient in 50 mM potassium phosphate, 1 mM EDTA, pH 7.5. Purified FtsZ, which eluted at ϳ0.4 M ammonium sulfate, was concentrated, desalted with a Fast Desalting column (5-ml Sephadex G-25 superfine, Amersham Biosciences) equilibrated with 20 mM Tris-HCl, 1 mM NaN 3 , 1 mM EDTA, pH 8.0, concentrated again, and stored at Ϫ80°C). FtsZ-His 6 and FtsZ-W319Y-His 6 were purified as controls of this method and had the same assembly properties under standard conditions (described under "Results") as those purified by affinity chromatography. FtsZ from E. coli was purified and assembled as described (12,21).
FtsZ(wt), FtsZ-His 6 , FtsZ-W319Y, and FtsZ-W319Y-His 6 had practically identical far UV circular dichroism spectra. The guanidinium chloride unfolding curves of the four proteins could be fitted by twostate transition models, with identical half denaturant concentrations (average, 3.03 Ϯ 0.10 M guanidinium chloride) and unfolding free energy changes (average, 11.6 Ϯ 2.0 kcal mol Ϫ1 ) within experimental error (for FtsZ-His 6 , see Ref. 21). These results indicated that neither modification, the addition of the His 6 tag or the W319Y point mutation, had any detectable effects on the average secondary structure and on the stability of FtsZ to chemical unfolding.
Assembly, GTPase, and Bound Nucleotide Assays-FtsZ was diluted (Ն20-fold) or chromatographically equilibrated into potassium-containing assembly buffer (50 mM Mes-KOH, 50 mM KCl, 1 mM EDTA, pH 6.5), or sodium-containing assembly buffer (50 mM Mes-NaOH, 50 mM NaCl, 1 mM EDTA, pH 6.5) at 30°(the FtsZ proteins with a six histidine tag had a tendency to precipitate in the cold), centrifuged for 10 min at 50,000 rpm in a Beckman TLA120.2 rotor to remove any aggregates, and its concentration was measured. Aliquots of FtsZ (typically 12.5 M) were placed into a fluorometer cuvette thermostatted at 55°C (solution temperatures were measured with a thermocouple), and its 350-nm light scattering was monitored at 90° (21). Assembly was typically started 5 min after by adding 6 mM MgCl 2 and 1 mM GTP (or 0.1 mM GMPCPP). Samples (20 l) for electron microscopy were taken directly from the solution.
The GTP and GMPCPP hydrolyzing activities of FtsZ solutions maintained at 55°C in a thermostatting block (Eppendorf Thermostat Plus) were measured by taking 20-l samples at several times, extracting the nucleotide with cold 0.5 N HClO 4 (with guanosine as an internal standard), followed by neutralization and ionic pair HPLC analysis of nucleotides with an octadecyl silica column as described previously (25). The nucleotides bound to FtsZ polymers were analyzed by sedimentation (6 min at 60,000 rpm in a prewarmed Beckman TL100 rotor), followed by pellet resuspension, nucleotide analysis, and FtsZ concentration measurement.
Electron Microscopy and Image Processing-Samples were adsorbed to grids on a hot plate or mildly fixed with 0.5% glutaraldehyde and subsequently stained with 2% uranyl acetate. Images were taken at various magnifications with JEOL 1200EX-II microscopes operated at 100 kV, and recorded on Kodak SO-163 film. In the case of the FtsZ sheets, micrographs were recorded with a nominal magnification of ϫ40,000 and the 3 best images, as judged by optical diffraction, digitized in a Zeiss SCAI scanner with a sampling window corresponding to 3.5 Å/pixel. Lattice refinement of the images was carried out using the X-windows-based graphical environment SPECTRA (26) and the subsequent image processing, including lattice unbending, was performed using the MRC image processing suite (27). Transfer function correction was carried out using the Integrated Crystallographic Environment (ICE, Ref. 28). Images were merged with the program ORIGTILT (27), using for that the crystallographic group p222 1 , which was suggested by the program ALLSPACE (29). In the case of the isolated filaments of FtsZ from M. jannaschii and from E. coli, images were taken at ϫ60,000 and digitized with a sampling window corresponding to 2.4 Å/pixel. 528 and 268 areas of the thick filaments were respectively selected so that 6 FtsZ monomers (3 of each of the 2 filaments) were present, and were subsequently aligned by cross-correlation free-pattern methods (30) and processed using the XMIPP software package (31).

RESULTS
Reversible Nucleotide-dependent Assembly of FtsZ from M. jannaschii-The polymerization of purified FtsZ was systematically investigated with light scattering at 90°and electron microscopy. Initial attempts to assemble FtsZ from M. jannaschii at moderate temperatures failed. However, increasing the temperature permitted the observation of reversible, GTP-Mg 2ϩ -dependent archaeal FtsZ assembly, similarly to mesophilic bacterial FtsZs at lower temperatures. This is probably related to the fact that this hyperthermophilic archaeon has an optimal growth temperature of 85°C (32). Fig. 1A (trace 1) shows the time course of assembly of FtsZ (wt, 12.5 M) initiated by addition of 1 mM GTP and 6 mM MgCl 2 in potassiumcontaining Mes assembly buffer, pH 6.5, at 55°C, which were chosen as standard conditions. Prior to nucleotide addition at time zero, the light scattering was negligible. Twenty seconds later (the shortest time recorded) most of the large scattering increase due to FtsZ assembly had taken place, and it gradually decayed afterward following the hydrolysis of GTP to GDP (the solid circles in Fig. 1A are HPLC measurements of the GTP concentration in the solution). Addition of more GTP produced a new cycle of assembly and disassembly; the FtsZ polymers completely disassembled upon GDP addition in less than 20 s. Polymers could be also cycled by cooling and heating the solution, or by chelation of Mg 2ϩ with EDTA followed by excess Mg 2ϩ addition (not shown). A very small light scattering increase was observed with Mg 2ϩ only, and an insignificant increase with GDP and Mg 2ϩ , or with GTP or GDP only (Fig.  1A), indicating that similarly large polymers did not assemble under these conditions. Substitution of GTP by the slowly hydrolyzable analogue GMPCPP permitted prolonged assembly (Fig. 1A) during Ն2 h. Decreasing the GMPCPP concentra-tion to 0.1 mM resulted in a shorter assembly, which dropped following hydrolysis of the analogue (Fig. 1A).
Polymerizing FtsZ hydrolyzed one GTP per ϳ10 s at pH 6.5 and 55°C (Table I). Increasing the Mg 2ϩ concentration decreased the rate of GTP hydrolysis and increased the time stability of the FtsZ polymers. Half disassembly (50% of the maximal scattering value) was reached in ϳ4 min with 3 mM MgCl 2 , whereas it typically took about 9 min with 6 mM MgCl 2 and 12 min with 10 mM. Interestingly, disassembly of FtsZ closely followed the consumption of GTP in each of these cases. Fig. 1B shows a plot of the light scattering versus the GDP/GTP ratio of the solution, measured in these experiments. The relative light scattering intensities (which started from different absolute values at each MgCl 2 concentration) drop practically together upon hydrolysis of GTP. Similar observations were made with FtsZ-His 6 , with the difference that the polymers were apparently stable for Ն60 min with 10 mM MgCl 2 .
FtsZ with GTP and Mg 2ϩ mainly formed filamentous polymers of widths usually between 120 and 300 Å (see Fig. 2A). FtsZ with GMPCPP and Mg 2ϩ formed polymers (Fig. 2C) as well that were typically wider (ϳ800 Å) than the GTP-induced polymers. Together with the large GTP and GMPCPP polymers, isolated ϳ60 Å wide FtsZ filaments, straight ( Fig. 2A) and curved (Fig. 2B), could be also observed. FtsZ at 80°C, close to the 85°C optimal growth temperature of M. jannaschii (32), assembled with GTP and Mg 2ϩ similarly at 55°C.
FtsZ-His 6 , which is FtsZ with a C-terminal Gly-Ser-His 6 extension, initially employed for purification and crystallization (19,24), assembled similarly to FtsZ under our standard conditions at 55°C (not shown). However, upon modification of these conditions, FtsZ-His 6 showed significant differences with FtsZ. These included (i) the assembly into helical tubes (  2D) with Ca 2ϩ (instead of Mg 2ϩ ) and also with Na ϩ (instead of K ϩ ), (ii) polymerization with Na ϩ without added nucleotide forming wide tubes (Fig. 2G), and (iii) formation of ordered sheets (Fig. 2E) upon assembly at 80°C with Mg 2ϩ (with GTP, GDP, or without added nucleotide). The above results indicated that the ␥ phosphate of the guanosine nucleotide and Mg 2ϩ promote FtsZ assembly; they suggested that nucleotide hydrolysis by the polymer results in its disassembly, and that the six histidine extension enhances assembly, favoring the formation of helical and laminar polymers instead of the filamentous polymers. The structures of the different polymers and of the FtsZ filaments will be addressed later.

Activation of FtsZ GTPase by Assembly in K ϩ -containing
Buffer-GTP-induced assembly of FtsZ into large light scattering polymers and significant GTP hydrolysis were found to take place above a critical FtsZ concentration (Cr Ӎ 2 M FtsZ). FtsZ assembly measured by sedimentation is cooperative and proceeds above a critical protein concentration in a nucleated condensation polymerization reaction. 2 Fig. 3 shows parallel measurements of GTP hydrolysis and light scattering at varying FtsZ concentration, indicating that the unassembled FtsZ 2 S. Huecas and J. M. Andreu, submitted for publication.  Table II  Nucleotide hydrolysis could be uncoupled from the assembly of FtsZ by replacing K ϩ by Na ϩ in the buffer, or with a point mutation inactivating the GTPase activity. Fig. 4 shows the time courses of assembly and nucleotide consumption in sodium-containing Mes assembly buffer at 55°C. The nucleotide hydrolysis rate decreased more than 100-fold with respect to the rate with K ϩ (Table I). Under these conditions FtsZ-His 6 (12.5 M) formed stable helical tubes (Fig. 2D) with GTP or GMPCPP plus Mg 2ϩ , and also with Mg 2ϩ only, but not with GDP plus Mg 2ϩ (since GDP is inhibitory, the polymer induction by Mg 2ϩ alone may be attributed to the residual GTP in purified FtsZ; see "Experimental Procedures"). The FtsZ-His 6 polymers assembled with GTP were found to contain 94% GTP and 6% GDP. Interestingly, addition of excess GDP to these nonhydrolyzing FtsZ-His 6 polymers in Na ϩ buffer resulted in their rapid disassembly (half-disassembly time, 40 s; Fig. 4B). FtsZ in Na ϩ buffer assembled into weakly light scattering stable polymers, which did not hydrolyze nucleotide but disassembled by GDP addition in Յ30 s (Fig. 4B).
Assembly of the GTPase-inactive Mutant FtsZ-W319Y-His 6 -FtsZ and FtsZ-His 6 assemble in a GTP and Mg 2ϩ -dependent fashion, forming polymers that can hydrolyze GTP, as shown above. The mutation of FtsZ-His 6 into FtsZ-W319Y-His 6, was chosen to be investigated due to the location of the single tryptophan (Trp-319) at the GTPase-activating interface of FtsZ, and the low natural frequency of the Trp 3 Tyr substitution at this position. This mutation inhibits the GTPase activity of FtsZ polymers and it favors the formation of stable ordered assemblies. Fig. 5 shows the time course of assembly of FtsZ-W319Y-His 6 . Assembly was induced by GTP plus Mg 2ϩ , and by Mg 2ϩ alone, but not by GDP and Mg 2ϩ . This is qualitatively similar to the results of inhibiting the GTPase by Na ϩ /K ϩ substitution (Fig. 4), except that GDP addition failed to reproducibly induce disassembly. The GTPase activity was similarly reduced, at least by two orders of magnitude (Table I). The Mg 2ϩ -induced assembly without added nucleotide (Fig. 5) may be explained by the residual GTP bound to purified FtsZ-W319Y-His 6 . The main structures formed by FtsZ-W319Y-His 6 where large ordered two-dimensional polymers made of filaments (Fig. 2E). Other structures formed by FtsZ-W319Y-His 6 , at 80°C, were rolled sheet polymers (Fig. 2H) in K ϩ -containing buffer without nucleotide or Mg 2ϩ , and also thick filamentous polymers formed with Mg 2ϩ plus GTP or GDP (Fig. 2F). Assembly of FtsZ-W319Y has only been observed at 80°C, forming thick polymers (Fig. 2F), indicating that the polymerization of FtsZ-W319Y-His 6 is facilitated by the six histidine extension. FtsZ-W319Y-His 6 coassembled with FtsZ-His 6 . A double mutant FtsZ-W319Y-T92W-His 6 (23) assembled poorly under these conditions; however it also coassembled with FtsZ-His 6 , stabilizing it against disassembly by GTP consumption, and forming similar laminar structures (Fig. 2E).
The Polymorphic Assembly Products of Purified FtsZ-The assembly products of purified FtsZ were different types of polymers, depending on the solution conditions, as indicated by Table II. Representative electron micrographs of each polymer type are shown in Fig. 2. For a short summary, FtsZ and FtsZ-His 6 under standard GTP-Mg 2ϩ -K ϩ assembly conditions formed filamentous polymers and isolated straight filaments ( Fig. 2A), isolated curved filaments (Fig. 2B), and they also formed wide filamentous polymers with GMPCPP (Fig. 2C). FtsZ-His 6 assembled into helical tubes (Fig. 2D) with Ca 2ϩ and also formed wide tubes with Na ϩ (Fig. 2G). The inactivated GTPase mutant FtsZ-W319Y-His 6 assembled into sheet polymers (Fig. 2E) and helical tubes (Fig. 2D) under standard conditions. Each of these polymers was observed in good yield and assembled in a reversible, biochemically regulated manner, including induction by nucleotide triphosphate and Mg 2ϩ at 55°C, and inhibition by GDP.
The images of sheets (Fig. 2E) gave similar diffraction patterns showing a tubulin-like axial spacing between FtsZ subunits in a unit cell of dimensions ϳ42 Å to ϳ140 Å (Fig. 2E). These sheets were readily obtained from FtsZ-W319Y-His 6. Their structural features are very similar to those of the minoritary Ca 2ϩ two-dimensional crystals and the GMPCPP sheets studied by Löwe and Amos (5,33). The helical tubes of FtsZ-His 6 have a diameter of ϳ320 Å and unwind in some segments revealing their helicity (Fig. 2D). Wide tubes (Fig.  2H) form under related conditions (Table II). The helical tubes appear closely related to the hollow helical tubes formed with Ca 2ϩ and GMPCPP (5,33). The isolated filaments are roughly 60 Å wide (Fig. 2B), and appear to be similar to the filaments typically formed by assembly of bacterial FtsZ from E. coli (12,21). We could not obtain diffraction patterns of the filamentous polymers ( Fig. 2A).
These results suggest that the different polymers may involve basically similar FtsZ filaments, with variations in the lateral filament interactions leading to the assembly of the filamentous, helical, sheet, or tube polymers. The fact that the wild-type FtsZ formed filamentous polymers instead of the large well-ordered sheet and helical tube polymers formed by FtsZ-His 6 and FtsZ-W319Y-His 6 , suggest that the two-dimensional crystallization is facilitated by the six histidine extension, which possibly makes interactions inducing the ordered lateral association of the FtsZ-His 6 filaments, whereas FtsZ(wt) filaments laterally associate in a more disordered fashion. Therefore it appears that the FtsZ-His 6 sheet and tubes contain additional features not present in the more natural polymers of FtsZ(wt). However, they proved useful to investigate the FtsZ filament structure.
FtsZ Double-stranded Filament Structure-The analysis of the images of selected single-layered ordered sheets generated by FtsZ-W319Y-His 6 (Fig. 6, A and B) revealed similar features to those described by Löwe and Amos (5) for the FtsZ-His 6 Ca 2ϩ -induced sheets, a series of filaments running along the sheet axis. The three-dimensional reconstruction carried out by these authors on the FtsZ-His 6 Ca 2ϩ -induced sheets showed that the filaments are formed by two parallel FtsZ protofilaments and that the thicker filaments interact with each other in an antiparallel fashion. The processing and merging of some of the ordered sheets obtained by us (Fig. 6A) suggested an identical arrangement (Fig. 6C). Comparison of phases of symmetry-related reflections (29) pointed, as in the case of the FtsZ-His 6 Ca 2ϩ -induced sheets, to p222 1 as the proper crystallographic group, and the images were combined accordingly. The projection map revealed the same antiparallel arrangement of thick filaments formed by two parallel protofilaments, with thin connections between neighbor thick filaments (arrow in Fig. 6C). Interestingly, when polymers of wild-type FtsZ were visualized at the electron microscope, a reasonable percentage of thick filaments, either individually or forming part of disordered sheets could be observed (Fig. 6D). The averaging of several hundred small areas of individual filaments generated an image (Fig. 6E), which reveals more clearly the double protofilament nature of the thick filaments, with the FtsZ monomers of each protofilament running in parallel. The polar appearance of the thick filament in the mutant FtsZ sheets is lost by the averaging procedure and the lack of visible connections in the wild-type FtsZ filaments. Whereas the longitudinal repeating distance of the protofilament in the crystal structure (Fig. 6C) and in isolated filaments (Fig. 6E) is almost identical (43 and 44 Å, respectively), there is an apparent discrepancy with respect to the width of the thick filament in both types of structures (57 versus 88 Å). However, this can be explained by the fact that the thick filament represented in Fig. 6C, according to the three-dimensional reconstruction of the FtsZ-His 6 Ca 2ϩ -induced sheets carried out by Löwe and Amos, is tilted (see Fig. 3B of Ref. 5) and therefore its projection is smaller. Finally, when the filaments formed by bacterial FtsZ from E. coli under our experimental conditions (Fig. 6F) were subjected to the same averaging procedure, an almost identical double-stranded filament image was obtained (Fig. 6G).

Dynamic Assembly of FtsZ from M. janaschii Is Induced by GTP and Mg 2ϩ at High Temperature, and It Activates GTP Hydrolysis Leading to Polymer
Disassembly-This work first established the conditions for reversible biochemically regulated assembly of archaeal FtsZ from M. jannaschii, and indicated that FtsZ is activated by GTP binding for Mg 2ϩ -induced assembly. Assembled FtsZ(wt) hydrolyzes one GTP every ϳ10 s (depending on conditions), and when GDP becomes predominant in the solution the polymers disassemble. This provides the more basic features for archaeal FtsZ polymer assembly/ disassembly dynamics, similarly to bacterial FtsZ (9). In addition to the known dependence of GTPase activity of bacterial FtsZ on protein concentration (15,34,35), our results with archaeal FtsZ most clearly indicate that the enhancement of the GTPase activity is cooperatively induced by the intermolecular contacts between the FtsZ molecules in large polymers, which were detected in the same experiment above a critical concentration of FtsZ (Fig. 3). The low GTPase values of unassembled FtsZ might be attributed to contacts in FtsZ oligomers (35) or to a basal activity of FtsZ monomers. Adding a Cterminal His 6 tag to FtsZ had little effect on its GTPase or assembly (monitored by light scattering and electron microscopy) under our standard conditions. However, it was shown to enhance assembly under other conditions, allowing polymeri- zation by Ca 2ϩ addition without added GTP, or in Na ϩ -containing buffer, or at 80°C, with the formation of helical tubes, ordered sheet, and tubes, instead of filamentous polymers (see "Results," Table II, and Fig. 5).
Currently available results do not clarify the mechanism of FtsZ polymer dynamics, that is, whether the hydrolyzed nucleotide exchange takes place in the FtsZ polymers or in rapidly assembling and disassembling FtsZ monomers. In the first case (nucleotide site exchangeable in the polymer; Ref. 14), the FtsZ polymers would tend to be continuously assembled in the GTPcontaining cytosol, and FtsZ polymer destabilizing proteins would be absolutely required for regulation of the Z-ring. In the second case (nucleotide site occluded by assembly; a GDP polymer possibly stabilized by a GTP cap, as in tubulin; Ref. 14)  A shows a micrograph of an ordered array of FtsZ-W319Y-His 6 whereas B shows its computed diffraction pattern and C a projection map generated using p222 1 symmetry. The rectangle in C indicates the unit cell of the crystal, which contains four FtsZ monomers, whereas the asterisk and the arrow indicate respectively to the connection between monomers belonging to the same thick filament or to an opposing one. D shows a gallery of individual thick filaments and E the average image obtained after processing 528 areas extracted from individual thick filaments. F shows a similar gallery of the products of assembly of bacterial FtsZ from E. coli (20 M FtsZ in 50 mM Mes-KOH, 50 mM KCl, 2 mM dithiothreitol, 10 mM MgCl 2 , and 1 mM GTP, pH 6.5, 30°C) and G the average image obtained from 268 areas from these filaments. The bar in A represents 1000 Å, and the bars in E and F 100 Å. individual polymers would be intrinsically unstable once their subunits have hydrolyzed GTP, and stabilizing factors would be required. Our finding of a mixture of GTP and GDP in the sedimented polymers of M. jannaschii FtsZ would argue against immediate GTP hydrolysis and a GDP-polymer model (17); if confirmed with a faster separation method, it would also argue against a GTP-polymer model (16), and would favor the possibility that FtsZ forms mixed polymers whose stability is related to the proportion of GTP-bound and GDP-bound subunits.
FtsZ Assembles without GTP Hydrolysis in Na ϩ -containing Buffer and Disassembles by GDP Addition-The results of replacing K ϩ by Na ϩ in the assembly buffer conclusively showed that GTP or GMPCPP hydrolysis is not necessary for FtsZ assembly. They also indicated that the six histidine extension in FtsZ-His 6 induces helical assembly, which is not observed with wild-type archaeal FtsZ. The GTPase activities of bacterial FtsZ from E. coli (36) and from the archaeon Haloferax volcanii (37) have a similar K ϩ cation requirement, whereas FtsZ from Azotobacter vinelandii and from Thermotoga maritima were reported equally active with Na ϩ and K ϩ (11) (in contrast with tubulin, which upon microtubule assembly hydrolyzes GMPCPP faster with Na ϩ than with K ϩ ; Ref. 38). These and other reports (8 -10, 15, 16, 20) showed the modulation of FtsZ assembly and GTPase by monovalent and divalent cations; however, when FtsZ assembly took place it entailed significant nucleotide hydrolysis. Our results with M. jannaschii FtsZ in Na ϩ -containing assembly buffer provide for the first time non-hydrolyzing polymers of wild-type FtsZ. Interestingly, when GDP is added to mimic GTP hydrolysis these kinetically stabilized archaeal FtsZ polymers rapidly disassemble (Fig. 4). This confirms the role of GDP in FtsZ disassembly dynamics, and intuitively favors the notion of an exchangeable nucleotide binding site in these polymers, which would not be expected to exchange their subunits in such a short time, or alternately, a very rapid endwise disassembly.
The W319Y Mutation Inactivates the GTPase and Kinetically Stabilizes the FtsZ Polymers-A number of mutations have been shown to inhibit the GTPase activity of bacterial FtsZ, including the temperature sensitive E. coli mutant ftsZ84 (39). Of special interest are the mutants in the longitudinal contact interface between FtsZ monomers, including the mutants at conserved loop T7, which has been proposed to come in close contact with the GTP bound to the next monomer down the filament (4,5). Aspartate 212 is an essential residue for the GTPase activity of E. coli FtsZ (40 -42). The corresponding residue Asp-238 (or one of the close residues Asn-233 or Asp-235) is thought to activate GTPase hydrolysis in M. jannaschii FtsZ, similarly to the putative activation of ␤-tubulin GTPase by residue Glu-254 of ␣-tubulin (4,5). The single tryptophan residue Trp-319 of FtsZ from M. jannaschii is located at the bottom association interface of FtsZ, less than 10 Å away from co-catalytic loop T7. The new mutant FtsZ-W319Y-His 6 assembled without hydrolyzing GTP. From 64 FtsZ sequences aligned from the SwissProt data base, the residues at the position corresponding to Trp-319 of M. jannaschii FtsZ were mainly Phe (25) and Trp (15), followed by Leu (6), Val (5), Ala (4), Ile (3, including Ile-294 of E. coli FtsZ), and less frequently Arg (2), His (1), or Tyr (Ref. 1; Borrelia burgdofei FtsZ), suggesting that an adventitious interaction of the Tyr hydroxyl group, or its reduced hydrophobicity, are not well tolerated by the functionality of FtsZ. Note that FtsZ-W319Y assembled poorly with respect to FtsZ, indicating that the mutation also affects in vitro FtsZ assembly, which is probably rescued by the His 6 tag in FtsZ-W319Y-His 6 .
The Basic Archaeal FtsZ Polymer Structure Is a Pair of Symmetric Tubulin-like Protofilaments-FtsZ, FtsZ-His 6 , and FtsZW319Y-His 6 assembled into polymorphic filamentous structures, suggesting a common type of filament with different lateral associations. Our results, obtained under biochemically regulated assembly conditions, confirm the basic doublestranded structure of FtsZ-His 6 filaments induced by Ca 2ϩ (5). The manual docking of the atomic structure of FtsZ from M. jannaschii into the three-dimensional reconstruction of the FtsZ protofilaments carried out by Löwe and Amos (5) suggests an arrangement of the FtsZ monomers within the protofilament similar to that of tubulin protofilaments. According to these authors, ␤-strand S3 is responsible of the interaction between protofilaments that form the thick filament (asterisk in Fig. 6C) whereas the C-terminal region (which is disordered in the x-ray crystal structure, and has an extra 8-residues fragment, including a six histidine tag) could bridge neighbor thick filaments (arrow in Fig. 6C). Since this extra region is not present in naturally occurring FtsZ, which generates laterally unordered filamentous polymers instead of ordered sheets, it is reasonable to suggest that these sheets are artificial and generated by the presence of the extra C-terminal residues. On the other hand it is likely that the interaction between two protofilaments is natural and gives rise to the thick filaments. This is confirmed by the novel image analysis of the filaments of wild-type FtsZ from M. jannaschii and also from E. coli (Fig. 6,  E and G), which indicate that many of them are similarly made of two parallel protofilaments with the same axial spacing between monomers. Whereas in vitro FtsZ polymers were reported to be made of one (15), two, or several protofilaments (9,12,16,20,21), image processing (Fig. 6) defines the doublestranded filament as a basic unit of FtsZ assembly.
The lateral interactions between two symmetric protofilaments of archaeal FtsZ are clearly different from those between microtubule protofilaments, as schematically depicted in Fig. 7.
In this new assembly model the two FtsZ protofilaments cannot use their common lateral bonding interface to further associate, whereas the same lateral interactions between tubulin protofilaments are known to propagate indefinitely until reaching cylindrical closure (43). In addition, neighbor microtubule protofilaments are staggered by 9D, whereas the symmetrical FtsZ protofilaments are not. The lateral association interfaces of tubulin map to its characteristic loop insertions that are absent from FtsZ, and which we have proposed to have evolved FIG. 7. A simplified geometrical comparison of the mode of assembly of FtsZ proposed in this work with the known assembly of tubulin. The filled circles indicate the position of the nucleotide and the letter C that of the C-terminal ends. FtsZ monomers assemble into a pair of symmetric protofilaments. Tubulin dimers assemble into similar protofilaments, which associate side to side by means of different interactions, until cylindrically closing into a microtubule.
with extended two-dimensional polymerization (21), a feature which has not been observed in spontaneous in vitro assembly of natural FtsZ. The common fold of tubulin and FtsZ is thus employed to construct apparently similar protofilaments, however, these protofilaments further associate to perform different functions. The microtubules of the eukaryotic mitotic spindle are numerous large stiff polymers, roughly perpendicular to the cell division plane, which push and pull chromosomes for DNA segregation. The Z-ring marks the cell division plane for prokaryotic cytokinesis and directs the formation of the septum, but has not been actually shown to perform any mechanical constriction work. It may be speculated that these different functions have evolved from a common ancestor, by means of two different modes of protofilament accretion, the robust tubular assembly of tubulin and a simple protofilament pair in the case of FtsZ.
It is possible that FtsZ polymers in bacteria share the double-stranded filament structure, and that the Z-ring may be made of a few double stranded filaments, which have resisted observation by electron microscopy. In the prokaryotic cytosol, FtsZ polymerization and lateral association of the FtsZ polymers is specifically modulated by interaction with the other cell division proteins (2,3). It can be calculated (8,18) from an estimate of the average number of FtsZ molecules per E. coli and the cell diameter (11), and the 43 Å axial spacing between FtsZ monomers (see "Results"), if only a part (ϳ30%) of FtsZ was incorporated into the Z-ring (6), that there might be as little as 3.5 continuous turns (on average) of FtsZ filament in a dividing cell. A similar calculation for B. subtilis cells (44) gives just a single functional FtsZ protofilament pair per Z-ring. Alternately, the Z-ring might be a scaffold made of Ն 10 FtsZ double-stranded filaments (similar to the wider filament in Fig.  5C), whose in vitro formation is favored by macromolecular crowding 2 (45), but more FtsZ molecules would be needed to form such a ribbon-like Z-ring.