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FtsZ is a prokaryotic homolog of tubulin and is a key molecule in bacterial cell division. FtsZ with bound GTP polymerizes into tubulin-like protofilaments. Upon polymerization, the T7 loop of one subunit is inserted into the nucleotide-binding pocket of the second subunit, which results in GTP hydrolysis. Thus, the T7 loop is important for both polymerization and hydrolysis in the tubulin/FtsZ family. Although x-ray crystallography revealed both straight and curved conformations of tubulin, only a curved structure was known for FtsZ. Recently, however, FtsZ from Staphylococcus aureus has been shown to have a very different conformation from the canonical FtsZ structure. The present study was performed to investigate the structure of FtsZ from Staphylococcus aureus by mutagenesis experiments; the effects of amino acid changes in the T7 loop on the structure as well as on GTPase activity were studied. These analyses indicated that FtsZ changes its conformation suitable for polymerization and GTP hydrolysis by movement between N- and C-subdomains via intermolecular interactions between bound nucleotide and residues in the T7 loop.
FtsZ is an essential bacterial cell division protein and is a prokaryotic homolog of the tubulin family (1, 2). FtsZ assembles into tubulin-like protofilaments by head to tail association (3) and forms during cell division a contractile ringlike structure known as the Z-ring at the midpoint of the cell anchored to the cytoplasmic membrane by several accessory proteins (2).
Tubulin is known to alternate between a curved conformation and a straight microtubule conformation depending on the bound nucleotide in the assembly/disassembly cycle. The structures of tubulin have been studied extensively (4,–10). In particular, the structures of the straight and curved protofilaments have both been determined by electron microscopy (7) and x-ray crystallography (8). In addition to bends at the subunit interface, the two subdomains within each tubulin subunit show marked rotations when changing from the straight to the curved protofilament conformation (9). A structure of capped tubulin dimers is now available (10), and this has a subdomain conformation identical to that in curved protofilaments, suggesting that the curved subdomain conformation is the relaxed state.
Protofilaments of FtsZ were also found to form straight and curved structures in relation to the bound nucleotide, as determined by electron microscopy (11, 12). A molecular dynamics simulation suggested that bending the H7 helix of FtsZ acts as a nucleotide-regulated molecular switch in the assembly/disassembly cycle of cell division (13). Escherichia coli FtsZ with GTP may switch the conformation in vitro, and flexibility between subdomains is important for GTPase activity in FtsZ from E. coli (14). The dynamics simulation also suggested that GTP-bound FtsZ dimer has a more rigid molecular formation than GDP-bound dimer, and the simulation predicted that the GDP-FtsZ filament is much more curved than the relatively straight GTP-FtsZ filament (15).
The enzymatic domain of FtsZ is known to act as a self-activating GTPase (16, 17). It is composed of two globular subdomains (N- and C-terminal subdomains) separated by a central core helix (H7 helix) and a synergy loop (T7 loop). There is a nucleotide-binding pocket in the N-terminal subdomain (residues 13–173). The C-terminal subdomain is likely a GTPase-activating subdomain (residues 223–310). For polymerization, the T7 loop in one subunit is inserted into the nucleotide-binding pocket of the next subunit (18). Mutation of Asn207, Asp209, and Asp212 in the T7 loop of FtsZ from E. coli severely affected GTP hydrolysis (19), suggesting that these catalytic residues in the T7 loop are poised to attack the γ-phosphate of GTP for GTP hydrolysis in the FtsZ polymer. In this way, GTPase activity is highly dependent on the polymerization of FtsZ (3, 19).
A number of structures of bacterial and archaeal FtsZs have been determined by x-ray crystallography (3, 20,–22). All of these structures were very similar, irrespective of the bound nucleotide, and the slight differences observed seemed to be related to interspecies differences (22). Recently, however, Staphylococcus aureus FtsZ (SaFtsZ)3 has been shown to have a markedly different structure by sliding/rotation between two subdomains with concomitant conformational change of the T7 loop (23). This structure seemed to be related to the polymerization state; the T7 loop of the “upper” subunit in SaFtsZ was inserted more deeply into the nucleotide-binding pocket of the “lower” subunit than any other structure determined to date. Therefore, the SaFtsZ structure has been interpreted as representing a straight protofilament conformation, whereas other structures are curved (and relaxed) conformations (23). This view is reinforced by the observation that SaFtsZ bound with PC190723, which inhibits GTPase activity by stabilizing the polymer formation (24), has the same conformation (23, 25, 26). Very recently Li et al. (27) showed that our FtsZ structure is consistent with their mutagenesis work. The rotation of the subdomains from curved FtsZ to straight FtsZ is in approximately the same direction as that of tubulin but larger in magnitude. A structural change induced by intermolecular interaction had been proposed to explain FtsZ assembly (28, 29).
In the present study, we analyzed the molecular characteristics of SaFtsZ by investigating the effects of the amino acid change in the T7 loop on the structure as well as on the GTPase activity. For this purpose, three mutants were prepared: a chimera mutant (T7Bs), the T7 loop of which has been replaced by that of Bacillus subtilis FtsZ (BsFtsZ), and two truncated mutants (ΔT7GAG and ΔT7GAN) in which the T7 loop was shortened. In addition, we also determined the co-crystallized structure of SaFtsZ-GTPγS. Here, we discuss these structural features in relation to the polymerization and GTPase activities of FtsZ.
Full-length (SaFtsZ) and enzymatic domain (residues 12–316, SaFtsZt) of FtsZ from S. aureus Mu50 were amplified and expressed previously (23). Mutant SaFtsZs were prepared using a Stratagene QuikChange mutagenesis kit (Agilent Technologies) with SaFtsZ expression vectors as a template. The T7 loop (VSGEV) of SaFtsZt was replaced with the T7 loop (TPGLI) of B. subtilis FtsZ (designated as T7Bs). Residues 204–208 (SGEVN) in the T7 loop of SaFtsZ were substituted for GAG and GAN (designated as ΔT7GAG and ΔT7GAN, respectively). The DNA sequences were confirmed using an ABI 310 Genetic Analyzer (Applied Biosystems).
Protein expression and purification of SaFtsZ mutants were performed as described previously (23). Purified proteins were concentrated to 5.0 mg/ml using Vivaspin 20–10K ultrafiltration devices (GE Healthcare) in storage buffer (50 mm Tris-HCl, pH 8.0, 300 mm NaCl).
Cell lysate of T7Bs in lysis buffer (50 mm Tris-HCl, pH 8.0, 300 mm NaCl, 10% (v/v) glycerol, 0.5 mg/ml lysozyme, and 0.1 mg/ml DNase I) was sonicated and then centrifuged at 40,000 × g for 30 min at 10 °C. The precipitate was resuspended in denaturing buffer (50 mm Tris-HCl, pH 8.0, 300 mm NaCl, 6 m guanidine hydrochloride) and dialyzed against 3 liters of dialysis buffer (50 mm Tris-HCl, pH 8.0, 300 mm NaCl, 10% (v/v) glycerol) three times to yield the refolded T7Bs. The dialyzed resuspension was centrifuged at 40,000 × g for 30 min at 10 °C. The purification procedures using chromatography were the same as described above (23). The refolded SaFtsZ was obtained according to the previously described procedure (23).
All crystals were obtained under the conditions shown in Table 1 and appeared after a few weeks at 20 °C. X-ray diffraction data of ΔT7GAG-GDP, ΔT7GAN-GTP, and refolded T7Bs were collected on beamline BL41XU of SPring-8 (Hyogo, Japan) under cryogenic conditions at −173 °C. These diffraction data were processed and scaled with either the HKL-2000 program package (30) (ΔT7GAG-GDP and T7Bs) or XDS (31) (ΔT7GAN-GTP). X-ray diffraction data of ΔT7GAN-GDP and refolded SaFtsZ-GTPγS were collected on Beamlines BL1A and BL17A of the Photon Factory (Tsukuba, Japan), respectively, under cryogenic conditions at −173 °C. Diffraction data of ΔT7GAN-GDP and SaFtsZ-GTPγS were processed and scaled with XDS (31). Molecular replacements of T7Bs, ΔT7GAN-GTP, and SaFtsZ-GTPγS were performed with 3VOA (SaFtsZt) (23) as the search model using Molrep in the CCP4 suite (32, 33). Molecular replacements of ΔT7GAG-GDP and ΔT7GAN-GDP were performed with 2VXY (B. subtilis FtsZ; BsFtsZ) (34) and ΔT7GAG-GDP as the search model using Molrep in the CCP4 suite, respectively. The structures were modified manually with Coot (35) and refined with Phenix (36).
The data collection and refinement statistics are summarized in Table 1. Structures were displayed using PyMOL (37). Coordinates and structure factors of T7Bs, ΔT7GAG-GDP, ΔT7GAN-GDP, ΔT7GAN-GTP, and SaFtsZ-GTPγS have been deposited in the RCSB Protein Data Bank under the accession numbers 3WGJ, 3WGK, 3WGL, 3WGM, and 3WGN, respectively.
SaFtsZ GTPase assay was performed as described previously (23). GTP was added to 5 μm proteins in reaction buffer (50 mm MOPS, pH 7.2, 5 mm MgCl2, 200 mm KCl) at a final concentration of 100 μm to start the reaction at 25 °C. Then 65 mm EDTA was added to the sample at various time points to stop the reaction. The samples were mixed with BIOMOL GREEN (BIOMOL Research Laboratories) and incubated at 25 °C for 30 min before measurement.
Although ΔT7GAG was crystallized in only one form, ΔT7GAN had several different crystal forms with different unit cell parameters (Table 2). Two of them were studied, and their structures were determined at resolutions of 3.07 and 2.10 Å, respectively (Table 1). ΔT7GAG and T7Bs were refined at resolutions of 2.80 and 2.18 Å, respectively (Table 1). The space groups of ΔT7GAG and two ΔT7GAN crystals were P21, whereas T7Bs crystal was P1. The crystals of all mutants contained two molecules in the asymmetric unit, and there were no significant differences in these two molecules in each crystal. All of these mutants crystallized as straight protofilaments forming longitudinal interactions (described in detail under “Molecular Interactions in the Crystal”). The electron density maps of T7Bs indicated the absence of bound nucleotide in the nucleotide-binding pocket of both independent molecules in the crystal, whereas a calcium ion was coordinated with the conserved residues in the T7 loop. Although the electron density maps of ΔT7GAG (ΔT7GAG-GDP) and one of ΔT7GAN (ΔT7GAN-GDP) crystals indicated the presence of GDP in the nucleotide-binding pocket, GTP and a magnesium ion were observed in the nucleotide-binding pocket of the other ΔT7GAN crystal (ΔT7GAN-GTP). All mutagenized residues were identified in the electron density maps.
Fig. 1 shows the crystal structures of all mutants superposed on both BsFtsZ (left panels) and SaFtsZ (right panels). All pictures in Fig. 1 were drawn after superposition of the N-terminal subdomains (residues 13–173). Although the N-terminal subdomains were superposed well in all mutants, the overall structures were considerably different (Fig. 1 and Table 3) because of the differences in relative orientation of the subdomains. The C-terminal subdomains of four mutants were moved downward (i.e., from the BsFtsZ type to SaFtsZ type) in the order of ΔT7GAN-GDP < ΔT7GAG-GDP < T7Bs < ΔT7GAN-GTP, as judged by the overlap with BsFtsZ or SaFtsZ (Fig. 1 and Table 3).
The conformation of T7Bs is shown in Fig. 1C. Although the amino acid sequence of the T7 loop in T7Bs was replaced by that of BsFtsZ from SaFtsZ, the overall conformation of T7Bs showed the same structural features as SaFtsZ (r.m.s.d. of 0.39 Å for backbone Cα atoms of C-terminal subdomains as in Table 3) and was different from that of BsFtsZ (r.m.s.d. of 7.54 Å). The main chain of the T7 loop in T7Bs also converged well with that in SaFtsZ (r.m.s.d. of 0.50 Å).
Fig. 1B shows structural comparisons of ΔT7GAG-GDP with BsFtsZ and SaFtsZ. Although the conformation of the N-terminal subdomain in ΔT7GAG-GDP was almost the same as those in the two FtsZs, the overall structure was not the same as either structure (Fig. 1B and Table 3) because of the differences in relative orientation of the subdomains. This is also the case for ΔT7GAN-GDP (Fig. 1A). There were almost no structural differences between ΔT7GAG-GDP and ΔT7GAN-GDP (r.m.s.d. of 0.74 Å; Table 3). The H6-H7 loop and T7 loop in both ΔT7GAG-GDP and ΔT7GAN-GDP were located in different positions from those in SaFtsZ but at similar positions to those in BsFtsZ (Fig. 1, A and B). The H7 helix in ΔT7GAG-GDP and ΔT7GAN-GDP was shifted by approximately one helical pitch in comparison with that in SaFtsZ (Fig. 1, A and B). To evaluate these changes in orientation, we used the DynDom program, which determines hinge axes by comparing two conformations (38). This program indicated that the subdomain orientation of ΔT7GAG-GDP and ΔT7GAN-GDP was also more similar to BsFtsZ than SaFtsZ (Table 3).
The structure of ΔT7GAN-GTP was different from any of these ΔΤ7 mutants bound with GDP. The H6-H7 loop and the T7 loop in ΔT7GAN-GTP were located in positions similar to those in SaFtsZ (Fig. 1D, right). The H7 helix in ΔT7GAN-GTP also showed the same shift as seen in SaFtsZ. However, the downshift of the C-terminal subdomain of ΔT7GAN-GTP from BsFtsZ structure was slightly greater than that in SaFtsZ (Fig. 1D, right panel, and Table 3).
To investigate the correlation between the monomer structure and molecular interactions in the polymer, we examined the crystal packing of T7Bs, ΔT7GAG-GDP, ΔT7GAN-GDP, and ΔT7GAN-GTP. SaFtsZ had a head to tail association with a molecule interval of 44.0 Å (23). T7Bs had the same symmetry association and molecular interval as seen in SaFtsZ (Table 1); two independent molecules were aligned on the a and b axes in its unit cell. The packing of ΔT7GAG-GDP, ΔT7GAN-GDP, and ΔT7GAN-GTP was formed by the head to tail association aligned on the a axis in their unit cells. The aligned distances in ΔT7GAG-GDP, ΔT7GAN-GDP, and ΔT7GAN-GTP were 44.3, 44.9, and 44.5 Å, respectively. The interactions between the aligned molecules of ΔT7GAN-GDP (and ΔT7GAG-GDP) were different from that in SaFtsZ, especially with regard to S9 strand and H10 helix in the C-terminal subdomain (Fig. 2, A and C). The H10 helix in the upper molecule of SaFtsZ was inserted into the cleft between the H6-H7 loop and the N-terminal subdomain in the lower molecule. The S9 strand of SaFtsZ also interacted with the cleft (Fig. 2C). The S9 strand and H10 helix in the upper molecule of ΔT7GAN-GTP was also located near the cleft in the lower molecule, and these interaction features were the same as those in SaFtsZ (Fig. 2, B and C). However, the S9 strand and H10 helix in the upper molecule of ΔT7GAN-GDP (and ΔT7GAG-GDP) were farther away from the cleft in the lower molecule than SaFtsZ because of the relative orientation differences (Fig. 2A). The interface area between the interaction molecules as calculated by PISA (39) was over 1230 Å2 for SaFtsZ (PDB code 3VOA), whereas those of ΔT7GAG-GDP and ΔT7GAN-GDP were under 810 and 740 Å2, respectively. The orientation change between N-terminal and C-terminal subdomains markedly diminished the molecular interactions. Although crystal packing of ΔT7GAN-GTP was more similar to that of SaFtsZ (Fig. 2, B and C), the interface area of ΔT7GAN-GTP was less than 1170 Å2. These results suggest that flipping the T7 loop out of the GTP-binding pocket also reduces the molecular interaction. Thus, the interactions between adjacent molecules in ΔT7mutant crystals were strongly correlated with the subdomain orientation in the monomer.
To clarify the characteristics of T7 loop mutants, we measured their GTPase activities (Fig. 3). The GTPase activity of T7Bs was similar to that of SaFtsZt. In contrast, GTPase activities of ΔT7GAG and ΔT7GAN were markedly reduced. In particular, it is noteworthy that ΔT7GAN, which retains all known catalytic residues, showed almost complete loss of GTPase activity. GTPase activity is highly dependent on the polymerization of FtsZ (3, 19). Thus, loss of GTPase activities in two ΔT7 mutants is likely to be due to defects in their polymerization activities.
Refolded SaFtsZ was co-crystallized with GTPγS, and its structure was determined at 2.61 Å (Fig. 4C and Table 1). The space group of GTPγS complex was P1. The crystal contained two molecules in the asymmetric unit. The electron density map of the crystal clearly showed the presence of GTPγS in the nucleotide-binding pocket of all independent molecules. However, the presence of a calcium ion could not be identified. The complex had a head to tail association with a molecular interval of 44.3 Å. There was no major conformational difference between the GTPγS complex and GDP complex determined previously (23) (r.m.s.d. of 0.59 Å; Fig. 4C). The packing interaction was also the same as seen in SaFtsZ-GDP (Fig. 4, A and B). The catalytic residues in the nucleotide-binding pocket, Asn208, Asp210, Asp213, and Asp46, were also at the same locations as SaFtsZ-GDP (Fig. 4, A and B). However, the electron densities of side chains of Asn44 and Gln48 in the pocket were too poor to determine their positions.
Although both straight and curved conformations of tubulin have been observed by x-ray crystallography, only a curved structure was known for FtsZ, possibly because it is more stable than any other state. However, FtsZ from S. aureus (SaFtsZ) has recently been shown to have a very different conformation from the canonical FtsZ structure determined previously (23). Briefly, the structure of SaFtsZ was different from the canonical FtsZ structure (e.g., BsFtsZ, PDB code 2RHL) in that the C-terminal subdomain slid down by approximately one helical pitch along the central H7 helix. This conformational change seemed to allow more intimate molecular association in the polymer and to enable the T7 loop of one molecule to intrude deeply into the GTPase pocket of the second molecule. The structure of FtsZ from Staphylococcus epidermidis deposited very recently (PDB code 4M8I) showed that its structure and longitudinal interactions were very similar to those of SaFtsZ. Possibly, some molecular interactions in these FtsZ crystals make the straight polymer type crystal more stable.
It has been suggested that the GTPase activity of FtsZ depends on the polymerization, and several residues in the T7 loop were proposed to be responsible not only for the GTPase activity but also for polymerization (19, 40). Therefore, in the present study, we investigated the effects of mutation of the T7 loop in SaFtsZ on the FtsZ structure as well as on the GTPase activity. The chimeric mutant T7Bs had the sequence of SaFtsZ in which the T7 loop sequence (VSGEV) was replaced by that of BsFtsZ (TPGLI). As expected, T7Bs retained the wild type level of GTPase activity (Fig. 3), and the structure was very similar to SaFtsZ rather than BsFtsZ (Fig. 1C), i.e., T7Bs crystallized in the form of assembled straight protofilaments, with a subdomain conformation very similar to that of polymerized SaFtsZ. These observations indicated that the species-specific difference in amino acid residues in T7 loop is not directly related to the change in FtsZ conformation and clearly excluded the possibility that the difference in amino sequence in the T7 loop was responsible for the different structure of SaFtsZ.
In contrast, the structure of ΔT7GAG-GDP was markedly altered to a structure similar to that of BsFtsZ (Fig. 1B and Table 3). The tip of the T7 loop in this mutant was shortened from the sequence SGEVN of SaFtsZ to GAG. Furthermore, ΔT7GAN-GDP, in which the important residue for GTPase activity (Asn208) is retained, also showed the same change (Fig. 1A and Table 3). Thus, the length of the T7 loop seems to be very important for the SaFtsZ type structure. Although ΔT7GAG-GDP and ΔT7GAN-GDP exhibited crystal packing with polymer-like molecular associations, their interactions were rather loose, consistent with the observation that they showed no GTPase activity. The T7 loop in the upper molecule was not deeply inserted into the nucleotide-binding pocket of the lower molecule (Fig. 2A). Moreover, the S9 strand and H10 helix in the upper molecule were distant from the C-terminal subdomain in the lower molecule because of the overall conformational change (Fig. 2A). Thus, the interaction surfaces between adjacent molecules in the crystals of these ΔT7 mutants were markedly reduced.
ΔT7GAN was also crystallized in GTP-bound form (ΔT7GAN-GTP). Interestingly, although the structure of ΔT7GAN-GDP was changed to the BsFtsZ type, ΔT7GAN-GTP showed features closer to SaFtsZ (Table 3). The H7 helix in ΔT7GAN-GTP had slid down by approximately one helical pitch with regard to the BsFtsZ type structure. Two residues in the H7 helix (Phe183 and Asp187) interacted with the guanine ring in GDP form, but only one residue (Phe183) retained stacking interactions with the guanine ring in ΔT7GAN-GTP. To our knowledge, this is the first observation of a nucleotide-dependent large conformational change of FtsZ; previous crystallographic studies did not detect any significant structural differences between FtsZ-GDP and FtsZ-GTP in the monomeric form (22). In the present study, we did not observe a major conformational difference between the GTPγS complex and the GDP complex determined previously (23) (r.m.s.d. of 0.59 Å; Fig. 4).The crystal structures of tubulin bound with GDP and GTP also showed no conformational change caused by nucleotide exchange (41).
Previous molecular dynamics simulation analysis in the monomeric form suggested that GDP to GTP exchange invokes a subtle conformational change in the nucleotide-binding pocket that tends to align the top portion of the H7 helix along the longitudinal axis of the protein (42). The conformational changes observed in ΔT7GAN-GDP and ΔT7GAN-GTP were, although more significant, somewhat in accordance with this prediction. The conformational change induced by GDP to GTP exchange should be attributed primarily to the γ-phosphate and Mg2+ ion in the nucleotide-binding pocket. However, the present crystal structure suggests that addition of γ-phosphate does not seem to have such a large effect directly on the FtsZ structure within one molecule. This large conformational change may be induced via interactions with the T7 loop of the second molecule.
The γ-phosphate interacted with the catalytic residues (Asp210 and Asp213) in the T7 loop and H8 helix of the upper molecule via water molecules in our GTP-bound model of SaFtsZ (23). This model was confirmed by the structure of SaFtsZ bound with GTPγS (Fig. 4A). The crystal packing of ΔT7GAN-GTP showed a similar head to tail association as seen in SaFtsZ. The crystal structure of ΔT7GAN-GTP also had interactions between γ-phosphate and the side chains of Asp210 and Asp213 via water molecules (Fig. 5B). These interactions between the T7 loop and H8 helix in the upper molecule and the residues around the nucleotide-binding pocket in the lower molecule were not detected in the crystal of ΔT7GAN-GDP (Figs. 2 and and5).5). Thus, it is likely that nucleotide exchange (addition of γ-phosphate) exerts an influence on the upper molecule via interaction with the T7 loop and induces the change in the subdomain orientation of the upper molecule, which leads to polymerization and GTP hydrolysis.
The structure of ΔT7GAN-GTP shows that the start and end residues of the T7 loop in ΔT7GAN-GTP were located at positions similar to those of the wild type. Despite the similar molecular associations in the polymer, the interactions at the tip of the T7 loop were very different between ΔT7GAN-GTP and the wild type (Figs. 4B and and55B). Because of the replacement of residues 204SGEVN208 with GAN at the tip of the T7 loop, the chain trace of the T7 loop was significantly changed, and Asn208 occupied a very different position in ΔT7GAN-GTP from that in the wild type (Figs. 4B and and5).5). It is likely that conserved residues, such as Asn208, Asp210, and Asp213, help the upper molecule interact with the bound γ-phosphate via the T7 loop and induce the conformational change, but because of truncation of the tip of the T7 loop, ΔT7GAN-GTP does not leave Asn208 at the proper position for GTP hydrolysis.
It may be worth mentioning that the downshift of C-terminal subdomain in ΔT7GAN-GTP was even larger than that in SaFtsZ (Fig. 1D and Table 3). The larger shift of the C-terminal subdomain of ΔT7GAN-GTP may be attributed to truncation of the tip of the T7 loop, which leaves more empty space at the region surrounded by the T7 loop and S9 strand. To maximize van der Waals interactions, the C-terminal subdomain may slide down further. This may reflect the intrinsic nature of the FtsZ molecule to adjust itself by changing the subdomain movement.
In this study, we observed several distinct conformations by changing the amino residues in the T7 loop of SaFtsZ. In the case of ΔT7GAN, we obtained two different conformations depending on the bound nucleotides. These results strongly suggested that the T7 loop triggers the whole structural change by rearranging the relative orientation between subdomains. It has been suggested that the T7 loop is a synergy loop with important roles not only for polymerization (19, 40) but also for the molecular switch between straight and curved conformations induced by binding of GTP (11, 12).
Recently, it was proposed that a hydrolysis-dependent conformational switch at the T3 loop of FtsZ leads to longitudinal bending between subunits (27). However, based on the results of the present study as well as on the structures deposited previously by other groups (PDB codes 2RHL, 2RHO, 2Q1X, and 2Q1Y) (43, 44), nucleotide exchange appears not to directly induce a structural change in the monomer, including the T3 loop. Rather, the present results suggest that the intermolecular interactions between bound GTP and the T7 loop of the second molecule induce the structural change in the molecule from the R (Relaxed) state to the T (Tense) state. This structural change allows more intimate interactions of the bound nucleotide with the catalytic residues of the second molecule in the straight polymer, resulting in GTP hydrolysis. After hydrolysis, the molecule returns to the R state and to the curved conformation (Fig. 6). The reorganization of the FtsZ subdomains is transmitted from one monomer to the other, and thus the intramolecular change may affect the intermolecular conformation as seen in tubulin polymers (9, 29).
We thank beamline staff of SPring-8 and Photon Factory for help in collecting x-ray diffraction data.
*This work was supported in part by the Project for Developing Innovation Systems of the Ministry of Education, Culture, Sports, Science, and Technology, Japan, and Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Young Scientists (B) 24770088.
3The abbreviations used are: