Crystal Structure of Ftsa from Staphylococcus Aureus

FEBS Letters 588 (2014) 1879–1885

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Crystal structure of FtsA from Staphylococcus aureus

Junso Fujita a, Yoko Maeda a, Chioko Nagao c, Yuko Tsuchiya c, Yuma Miyazaki a, Mika Hirose b, ⇑ Eiichi Mizohata a, Yoshimi Matsumoto d, Tsuyoshi Inoue a, Kenji Mizuguchi c, Hiroyoshi Matsumura a, a Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan b Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan c National Institute of Biomedical Innovation, 7-6-8, Saito-Asagi, Ibaraki, Osaka 567-0085, Japan d Laboratory of Microbiology and Infectious Diseases, Institute of Scientiﬁc and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan article info abstract

Article history: The bacterial cell-division protein FtsA anchors FtsZ to the cytoplasmic membrane. But how FtsA Received 5 February 2014 and FtsZ interact during membrane division remains obscure. We have solved 2.2 Å resolution crys- Revised 2 April 2014 tal structure for FtsA from Staphylococcus aureus. In the crystals, SaFtsA molecules within the dimer Accepted 2 April 2014 units are twisted, in contrast to the straight ﬁlament of FtsA from Thermotoga maritima, and the Available online 18 April 2014 half of S12–S13 hairpin regions are disordered. We conﬁrmed that SaFtsZ and SaFtsA associate Edited by Kaspar Locher in vitro, and found that SaFtsZ GTPase activity is enhanced by interaction with SaFtsA.

Structured summary of protein interactions: Keywords: Bacterial divisome SaFtsA and SaFtsZ bind by comigration in non denaturing gel electrophoresis (View interaction) Staphylococcus aureus SaFtsZ and SaFtsA bind by molecular sieving (View interaction) FtsA SaFtsA and SaFtsA bind by x-ray crystallography (View interaction) FtsZ Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction FtsZ and FtsA tend to form filamentous structures [7–9]. FtsZ forms straight protofilaments in the presence of GTP, thereby During bacterial cell division, more than 20 proteins constitute forming a GTPase active site between two FtsZ molecules. Hydro- a multi-protein complex called the divisome at the midcell [1]. lysis of GTP to GDP leads straight-to-curved conformational change FtsZ, a divisome protein, assembles to form the ring-like structure, of FtsZ protofilaments [9]. Crystal structures of a mesophilic and a the Z-ring, that allows the daughter cells to physically separate [2]. thermophilic FtsZ have been solved [10], and the recent crystallo- Polymerized FtsZ is tethered to the cytoplasmic membrane by FtsA graphic study of Staphylococcus aureus FtsZ (SaFtsZ) has proposed through its C-terminal membrane-targeting sequence (MTS) [3]. the mechanism how the straight-to-curved conformational change Optical microscopic studies have indicated that yellow fluorescent of FtsZ polymers is coupled to its GTPase activity [11,12]. protein (YFP) and MTS-fused FtsZ (FtsZ–YFP–MTS) dents a lipo- FtsA belongs to the actin/MreB family of proteins, and actin/ some membrane, but does not cause the liposome to divide [4], MreB possess ATPase activity coupled to their polymerization whereas the combination of FtsZ–YFP and FtsA⁄ (an FtsA gain-of- [13,14]. Although FtsA forms protofilaments similar to those of function mutant [5]) causes the formation of a continuous septum actin and MreB [7,8], the role of its ATPase activity remains to be in a GTP/ATP-dependent manner [6]. Therefore, FtsA appears to clarified. ATPase activity has been detected in vitro for Bacillus tether FtsZ to the membrane and may be necessary for down- subtilis [15] and Pseudomonas aeruginosa FtsAs [16], but it has not stream cell division. Both FtsZ and FtsA are essential for cell divi- been found for FtsA from Streptococcus pneumoniae [7], Escherichia sion and viability and they are highly conserved in many coli [17] and Thermotoga maritima [8]. Recent microscopic study bacterial species, making them attractive targets for the develop- shows that FtsZ and FtsA self-organize into a rapidly reorganizing ment of antibacterial agents. filament network [18], but understanding of the molecular function of FtsA during cell division has been hampered by the limited structural information available for a mesophilic FtsA; the only crystal structure available is that of the FtsA from the thermophile ⇑ Corresponding author. Fax: +81 668797409. T. maritima (TmFtsA) [8,19]. Therefore, a three-dimensional E-mail address: [email protected] (H. Matsumura). structure for a mesophilic FtsA is needed, but, until this report, http://dx.doi.org/10.1016/j.febslet.2014.04.008 0014-5793/Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. 1880 J. Fujita et al. / FEBS Letters 588 (2014) 1879–1885 difficulties had been encountered when preparation of a meso- Table 1 philic FtsA was attempted, e.g., refolding of P. aeruginosa and Data collection and refinement statistics. E. coli FtsA from inclusion bodies [16,17] and the inability to crys- Data set SaFtsA (AMPPNP) SaFtsA (ATP) SaFtsA (SeMet) tallize the folded product. PDB code 3WQT 3WQU We previously reported that full-length FtsA from mesophilic X-ray source Photon factory SPring-8 SPring-8 methicillin-resistant S. aureus (SaFtsA) could be purified in a trac- BL1A BL44XU BL44XU table form and crystallized [20]. Herein, we report the crystal Space group P21 P21 P21 Unit-cell parameters structure of SaFtsA-b-c-imidoadenosine 50-phosphate (AMPPNP; a (Å) 75.26 82.53 74.54 a non-hydrolyzable ATP analog) complex. In the crystal, the SaFtsA b (Å) 102.74 122.02 101.87 molecules stack head to tail forming a continuous filament, as does c (Å) 105.86 107.00 105.37 crystalline TmFtsA and TmMreB. However, SaFtsA dimer in the fil- b (°) 96.54 95.66 96.23 ament is twisted, and the torsion angles of the neighboring dimers Wavelength (Å) 1.00 0.900 0.979 Resolution (Å) 38.0–2.2 39.7–2.8 50.0–2.7 within the asymmetric unit are different. In addition, we show that (2.24–2.20)a (2.85–2.80)a (2.75–2.70)a SaFtsZ and SaFtsA form a complex in vitro and that SaFtsZ GTPase Total reflections 234088 196794 334143 activity is promoted by its interaction with SaFtsA, which sheds Unique reflections 78940 47837 84991 light on the coordinated regulation of FtsA and FtsZ during cell Completeness (%) 97.4 (97.4) 92.3 (86.5) 100.0 (100.0) R-merge (%)b 6.7 (39.6) 7.5 (59.3) 10.0 (58.5) division. I/r 18.6 (2.1) 18.7 (2.2) 14.7 (1.6) Phasing 2. Materials and methods No. of sites 17 FOMc 0.32 2.1. Protein expression and purification Refinement Resolution 38.0–2.2 39.7–2.8 SaFtsA (UniProt ID: Q6GHQ0) was expressed and purified as Rwork/Rfree 0.234/0.289 0.207/0.254 described [20]. SaFtsZ (UniProt ID: Q6GHP9) was cloned, No. of atoms Protein 11051 11487 expressed, and purified as reported for SaFtsA except that the clon- Ligand 139 128 0 ing primers were: ftsZ-for (5 -GCCATATGTTAGAATTTGAACAAGG Water 428 48 ATTTAATC-30) and ftsZ-rev (50-GCGGATCCTTAACGTCTTGTTCTTCT R.m.s. deviationd TGAACG-30). Selenomethionine-labeled SaFtsA (SeMet-SaFtsA) Bond length (Å) 0.007 0.009 was expressed and purified with the same protocols as wild-type Bond angles (°) 1.2 1.3 a (WT) SaFtsA, except that the expression system was E. coli B834 Values in parenthesesP are for theP highest resolution shells. (DE3) (Invitrogen) and that the cells were cultured in LeMaster b R-mergeðIÞ¼ j IðkÞ < I >j = IðkÞ, where I (k) is the value of the k th medium, 50 mg/L SeMet (QIAGEN), 100 lg/ml ampicillin (Wako), measurement of the intensity of a reflection, is the mean value of the intensity of that reflection and the summation is over all measurement. 5 ml/L vitamin solution as reported [21,22]. As assessed by c FOM – figure of merit = |F(hkl)best|/|F(hkl)|;F(hkl)best = RaP(a)Fhkl(a)/RaP(a). matrix-assisted laser desorption/ionization-time of flight mass d R.m.s.deviation – root-mean-square deviation. spectrometry, six of the possible seven methionines are present in SeMet-SaFtsA (SaFtsA: observed, 54679 Da; calculated, 54703 Da; SeMet-SaFtsA: observed, 54962 Da; full substitution: found by SHELXD [24] using the anomalous signals in the SeMet- calculated, 55031 Da). Site-directed mutagenesis of SaFtsZ SaFtsA peak datasets. Initial phases were calculated and refined (D210A) was performed by inverse-PCR using KOD-plus (TOYOBO) using SHELXE [24] and the graphical interface HKL2MAP [25]. and the primer 50-GAAGTAAACTTAGcaTTTGCAGACGTTAAGAC-30. Model refinement used CNS [26] and PHENIX [27], with manual (The changed nucleotides are shown in small letters, and the inspection and modification in conjunction with the CCP4 program codons corresponding to the amino acid residues to be changed COOT [28]. Phasing and refinement statistics are given in Table 1. are underlined). SaFtsZ D210A was expressed and purified with PROCHECK [29] indicated that >85% of residue /–u values for each the same protocols as WT SaFtsZ. All the proteins have N-terminal of structures are in the most favored regions of the corresponding extra residues of MNHKVHHHHHHIEGRH. Ramachandran plots. Chain interactions were assessed at the PISA server [30], and interface interactions were identified by LIGPLOT 2.2. Crystallization, data collection and structure determination [31]. Figures were prepared using Pymol (www.pymol.org) and ESPript [32]. The final atomic coordinates and structure factor SaFtsA was crystallized in the presence of AMPPNP as reported amplitudes (PDB codes 3WQT for with AMPPNP and 3WQU for [20]. SaFtsA was also crystallized in the presence of ADP by mixing with ATP) have been deposited in the Worldwide Protein Data an equal volume (1 ll) of protein solution (10 mg/ml) containing Bank (wwPDB; http://www.wwpdb.org), and the Protein Data

1 mM ADP and 2 mM MgCl2 with 0.2 M lithium nitrate, 20% (w/ Bank Japan at the Institute for Protein Research in Osaka University v) PEG3350, 0.1 M sodium cacodylate, pH 7.0 (reservoir solution) (PDBj; http://www.pdbj.org/). and equilibrating against 1 ml of the reservoir solution. The crystallization conditions for SeMet-SaFtsA were the same as those 2.3. GTPase/ATPase activity assay for crystallization of SaFtsA in the presence of AMPPNP. Crystals were individually mounted in a loop and then flash frozen in a GTPase or ATPase activity was measured by quantification of stream of nitrogen at 100 K. Synchrotron-radiation diffraction data free inorganic phosphate using Malachite Green Phosphate Assay were collected at 100 K at the SPring-8 BL44XU and Photon Factory kit reagents (BioAssay Systems), according to manufacturer’s BL-1A beamlines (Japan). The diffraction datasets were processed instruction. The reaction mixture (final volume 200 ll) contained using HKL2000 [23]. Data-collection statistics are summarized in 50 mM MOPS, pH 7.2, 5 mM MgCl2, 200 mM KCl, 0–1500 lM GTP Table 1. or ATP and was incubated at 37 °C for 15 min. The reaction mixture The structure of SaFtsA was determined by the single-wave- (32 ll) was diluted 25-fold with 50 mM MOPS, pH 7.2. The pre- length anomalous diffraction method using SeMet-labeled-SaFtsA. pared working reagent (200 ll) was added, and the mixture was Of the 24 possible selenium atoms in an asymmetric unit, 17 were incubated at 20 °C for 30 min. The absorbance at 620 nm was J. Fujita et al. / FEBS Letters 588 (2014) 1879–1885 1881 measured using a UV-2550 UV–Vis spectrophotometer (Shima- dzu), and phosphate concentration is calculated from phosphate standard curve by a blank subtraction. The phosphate concentration shown in Fig. 5 is multiplied by the dilution factor.

3. Results and discussion

3.1. Crystal structure of SaFtsA

Initial phases were obtained by single-wavelength anomalous diffraction phasing of the SeMet-FtsA data, and the structure of the SaFtsA-AMPPNP complex was resolved to 2.2 Å and refined to crystallographic R factor of 23.4% (Rfree = 28.9%, Table 1). The over- all structure of SaFtsA resembles that of TmFtsA (see below) [8,19] and shows the canonical actin-like fold (Fig. 1A). The SaFtsA mono- mer contains four domains, 1A, 1C, 2A, and 2B, as does the TmFtsA structure. The electron density map clearly shows that AMPPNP (or ATP, see Supplementary Information) is bound in the pocket formed by domains 1A, 2A, and 2B (Fig. 1B). The AMPPNP adenine ring is stabilized by the side chain of His255 via a p–p interaction. The side chains of Glu251, Lys254, and His255 are responsible for ribose stabilization. Two loops participate in the binding of the phosphates: the S1–S2 loop, found between b-strands 1 and 2, (residues 13–17) and the S9–S10 loop, found between b-strands 9 and 10 (residues 209–211). In addition, several water molecules form a hydrogen bond network in the ATP-binding site. Two of these water molecules (labeled W1 and W2) are located 4.5 and 4.8 Å from the AMPPNP Pc, and the b-c-bridging oxygen-Pc-water angles are 139° and 133°, respectively. In TmFtsA structures, the water molecule corresponding to W1 is located 4.3–4.6 Å from the Pc and the angle is 113–135°, and W2 was structurally replaced by the side chain of Ser84. These distances and angles indicate that the waters are not poised for an in-line nucleo- philic-attack on the ATP c-phosphate. Indeed, SaFtsA has very weak ATPase activity (see below). However, other biochemical Fig. 2. Crystal packing of SaFtsA. The crystallographic asymmetric unit contains studies have suggested that FtsA may exhibit actin-like ATPase four molecules, and the A, B and C, D molecules stack head to tail in the opposite directions to form two antiparallel filamentous structures. The dimer units (A, B and activity [15,16]. We favor the idea that the cell division proteins, C, D) are related by a 106 Å translations, but the two molecules in the units are which interact with FtsA might induce a structural change in FtsA twisted 28° (A, B) and 17° (C, D). The 1A–1C and 2B–2A interfaces are shown in red that results in an increase in its ATPase activity during cell division. and blue circles, respectively. The S12–S13 hairpin region is disordered only in the B and D chains (dashed circles). 3.2. Interactions between neighboring SaFtsA molecules head to tail in the crystal, thereby forming two antiparallel fila- The crystallographic asymmetric unit contains four SaFtsA mol- mentous structures (Fig. 2 and Supplementary Fig. S1). The A, B ecules (chains A, B, C, and D). The A, B and C, D molecules stack and C, D units are related by a 106 Å translation, but the relative

Fig. 1. Crystal structure of SaFtsA. (A) SaFtsA has the same domain architecture as TmFtsA; domains 1A, 1C, 2A, and 2B are colored in blue, yellow, red, and green, respectively. The S12–S13 hairpin region is colored in gray. (B) Fo–Fc omit map (contoured at 3.5 r as a blue grid) clearly shows the presence of AMPPNP in active site. Electron densities of low-occupancy W1 and W2 are contoured at 3.0 r as a purple grid. 1882 J. Fujita et al. / FEBS Letters 588 (2014) 1879–1885

Fig. 3. Structure comparison of the SaFtsA and the TmFtsA–FtsZ peptide complex (PDB code: 4A2A). (A) View rotated 180° around the vertical axis in Fig. 1. SaFtsA (yellow) has the canonical actin-like fold as does TmFtsA (blue; rmsd = 3.0 Å for 336 Ca atoms). The TmFtsZ peptide (red) binds near the TmFtsA S12–S13 hairpin. (B) Few interactions are seen between the S12–S13 hairpin and the underlying helices in the SaFtsA structure. (C) Many interactions are seen for the TmFtsA S12–S13 hairpin and the underlying helices, which decrease the mobility of the S12–S13 hairpin. The red dot indicates a water molecule which participates in the interactions. (D) Structure comparison of SaFtsA dimer unit (A, B, yellow) and that of TmFtsA (PDB code: 4A2B, blue). When the lower molecules are superimposed, the upper molecules are related by 28° rotation around the vertical axis. J. Fujita et al. / FEBS Letters 588 (2014) 1879–1885 1883 arrangements of the two molecules within the filaments are differ- direction (Fig. 4). This difference is likely caused by domain-swap- ent; the two molecules in the dimer units are twisted 28° (A, B) and ping and topological difference between 1C of FtsA and 1B of MreB, 17° (C, D). as described previously [8]. TmFtsA filaments orient in the same The interaction between two neighboring molecules in the fila- direction as SaFtsA, but SaFtsA dimer in the filaments are twisted, ments is mediated by two interfaces: 1A–1C and 2B–2A interfaces in contrast to straight TmFtsA filament in the crystals (Fig. 3D). To (Fig. 2, red and blue circles, respectively). The 1A–1C interface has a clarify whether this twist is required for functions of SaFtsA, fur- relatively large area (564 Å2) compared with the 2B–2A interface ther structural study would be needed. (174 Å2). The 1A–1C interface is stabilized by hydrophobic and polar interactions, which are essentially conserved in the A, B 3.3. GTPase assay of SaFtsZ–FtsA complex and C, D interfaces (Supplementary Fig. S2). This interface contains Leu145, which is equivalent to Met147 in B. subtilis FtsA (Supple- Complex formation by SaFtsZ and SaFtsA was confirmed by two mentary Fig. S3). When Met147 is mutated to a Glu, polymeriza- methods: size-exclusion chromatography and Native-PAGE (Sup- tion of FtsA does not occur [8]. The 2B–2A interactions are plementary Figs. S5 and S6). We then performed preliminary mediated by a few residues in the S12–S13 hairpin loop, loop GTPase assay using the malachite green kit to investigate the H8–S14, and loop H10–S15, but a variety of interactions are effects of complex formation on the GTPase activity of SaFtsZ. At observed at the A, B and C, D interfaces (Supplementary Fig. S2). 5 and 10 lM free SaFtsZ exhibits very little GTPase activity. When The S12–S13 hairpin is structurally ordered in A and C, whereas complexed with SaFtsA, at concentrations of 5 lM SaFtsZ and 5 lM it is disordered in B and D (Fig. 2, dashed circles). Normal mode SaFtsA, or 10 lM SaFtsZ and 2.5 lM SaFtsA, GTP is hydrolyzed inef- analysis of SaFtsA also suggests that the atoms in the S12–S13 hair- ficiently, but in mixtures containing 10 lM SaFtsZ and 10 lM SaF- pin in the 2B domain with increased fluctuations (Supplementary tsA, or 10 lM SaFtsZ and 5 lM SaFtsA, GTPase activity is much Fig. S4). The mutation point of FtsA⁄ (R286W gain-of-function more pronounced. A SaFtsZ mutant (D210A) lacking the GTPase mutant of EcFtsA) is located in the S12–S13 hairpin (Supplemen- activity did not show increased GTPase activity in the presence tary Fig. S3). Furthermore, the S13–H8 region of SaFtsA appears of SaFtsA (Fig. 5) [11]. to bind the C-terminal peptide of SaFtsZ, judging from the struc- Optical microscopic studies have indicated that MTS-fused FtsZ ture of the TmFtsA–FtsZ peptide complex (Fig. 3A). The SaFtsA only dents a liposome membrane [4], whereas the combination of S12–S13 hairpin is structurally distinct from those in the TmFtsA FtsZ and FtsA⁄ causes the formation of a continuous septum [6]; crystals [8,19]. Previous crystallographic studies of TmFtsA showed therefore, FtsA not only tethers FtsZ to the cytoplasmic membrane that no structural differences in the S12–S13 hairpin were found in but also appears to have a direct role in cell division. Based on our the different crystal forms, probably because the TmFtsA S12–S13 results, we suggest that SaFtsA regulates the GTPase activity of SaF- hairpin is stabilized by more hydrogen bonds and salt-bridges than tsZ. During cell division, the localization and the accumulation of SaFtsA (Fig. 3B and C). FtsZ caused by FtsA at the cytoplasmic membrane may facilitate Similar to TmFtsA, SaFtsA has the domain swapped architecture FtsZ polymerization and formation of the Z-ring. Once FtsZ forms compared to TmMreB (Fig. 1A) [8,33], and both FtsA and MreB mol- protofilaments, an increase in its GTPase activity induced by inter- ecules are translationally aligned to form filament-like structures action with FtsA might allow the filaments to curve. Such a in the crystals [34]. FtsA and MreB are able to bind the membrane. dynamic event might be needed to complete membrane division. Thus, FtsA and MreB share structural and functional properties; we However, in E. coli, FtsA does not affect FtsZ GTPase activity therefore compared the filamentous structures of SaFtsA with [18,35]. As mentioned above, FtsA has not yet been characterized TmMreB. Although the membrane-interaction sites are closely well, and we cannot exclude the possibility that FtsA from different located, the filaments of SaFtsA and TmMreB orient in a different species shows different behaviors. Furthermore, how SaFtsA

Fig. 4. Structure comparison of SaFtsA (yellow) and TmMreB (cyan) ﬁlaments. The left view is from the same direction as Fig. 2 left panel. When superimposed on the monomeric structure, the two ﬁlaments orient in a different direction. The last visible C-terminal residue (Asp377) for SaFtsA and membrane insertion loop (Leu93/Phe94) for TmMreB are indicated as blue and red circles, respectively. 1884 J. Fujita et al. / FEBS Letters 588 (2014) 1879–1885

Fig. 5. GTPase activity assay of the SaFtsZ–FtsA complex. FtsZ GTPase activity is enhanced when the FtsZ concentration is >5 lM and the molar ratio of FtsZ to FtsA is 62:1. A SaFtsZ mutant (D210A) lacking the GTPase activity showed no enhancement. The bars represent the standard errors of three independent experiments. enhances the GTPase activity of SaFtsZ is not yet understood. One [7] Lara, B., Rico, A.I., Petruzzelli, S., Santona, A., Dumas, J., Biton, J., Vicente, M., possibility is that the dynamics (association/dissociation and/or Mingorance, J. and Massidda, O. (2005) Cell division in cocci: localization and properties of the Streptococcus pneumoniae FtsA protein. Mol. Microbiol. 55, straight-to-curved conformational changes) of FtsZ molecules dur- 699–711. ing protofilament formation are affected by FtsA binding, thereby [8] Szwedziak, P., Wang, Q., Freund, S.M. and Lowe, J. (2012) FtsA forms actin-like elevating the GTPase activity. Structural elucidation of a full-length protofilaments. EMBO J. 31, 2249–2260. [9] Erickson, H.P., Anderson, D.E. and Osawa, M. (2010) FtsZ in bacterial FtsZ–FtsA complex may solve these questions. cytokinesis: cytoskeleton and force generator all in one. Microbiol. Mol. Biol. Rev. 74, 504–528. [10] Lutkenhaus, J., Pichoff, S. and Du, S. (2012) Bacterial cytokinesis: from Z ring to Acknowledgements divisome. Cytoskeleton 69, 778–790. [11] Matsui, T., Yamane, J., Mogi, N., Yamaguchi, H., Takemoto, H., Yao, M. and Tanaka, I. (2012) Structural reorganization of the bacterial cell-division We thank the staff of the SPring-8 BL44XU beamline, and the protein FtsZ from Staphylococcus aureus. Acta Crystallogr. D Biol. Crystallogr. Photon Factory BL-1A beamline, for help with collecting X-ray data. 68, 1175–1188. [12] Matsui, T., Han, X., Yu, J., Yao, M. and Tanaka, I. (2013) Structural change in This work was supported by the Knowledge Cluster Initiative pro- FtsZ induced by intermolecular interactions between bound GTP and the T7 moted by the Ministry of Education, Culture, Sports, Science, and in loop. J. Biol. Chem. 289, 3501–3509. part supported by Grants-in-Aid for Scientific Research (25121719, [13] Bork, P., Sander, C. and Valencia, A. (1992) An ATPase domain common to prokaryotic cell cycle proteins, sugar kinases, actin, and hsp70 heat shock 25440022 and 26102526 to H.M.; 24109017 to T.I.; 25430186 and proteins. Proc. Natl. Acad. Sci. U.S.A. 89, 7290–7294. 25293079 to K.M.), the Japan Society for the Promotion of Science [14] Esue, O., Wirtz, D. and Tseng, Y. (2006) GTPase activity, structure, and (JSPS) through the ‘‘Funding Program for World-Leading Innovative mechanical properties of filaments assembled from bacterial cytoskeleton R&D on Science and Technology (FIRST Program),’’ initiated by the protein MreB. J. Bacteriol. 188, 968–976. [15] Feucht, A., Lucet, I., Yudkin, M.D. and Errington, J. (2001) Cytological and Council for Science and Technology Policy (CSTP) to T.I, and the biochemical characterization of the FtsA cell division protein of Bacillus Platform for drug discovery, informatics and structural life science subtilis. Mol. Microbiol. 40, 115–125. (to H.M.) from the Ministry of Education, Culture, Sports, Science. [16] Paradis-Bleau, C., Sanschagrin, F. and Levesque, R.C. (2005) Peptide inhibitors of the essential cell division protein FtsA. Protein Eng. Des. Sel. 18, 85–91. This work has been performed under the approval of the Photon [17] Martos, A., Monterroso, B., Zorrilla, S., Reija, B., Alfonso, C., Mingorance, J., Factory and SPring-8 Program Advisory Committee (Proposal Rivas, G. and Jimenez, M. (2012) Isolation, characterization and lipid-binding Nos. 2013G148, 2012G533, 2012B6640, 2013A6848). properties of the recalcitrant FtsA division protein from Escherichia coli. PloS One 7, e39829. [18] Loose, M. and Mitchison, T.J. (2014) The bacterial cell division proteins FtsA Appendix A. Supplementary data and FtsZ self-organize into dynamic cytoskeletal patterns. Nat. Cell Biol. 16, 38–46. [19] van den Ent, F. and Lowe, J. (2000) Crystal structure of the cell division protein Supplementary data associated with this article can be found, FtsA from Thermotoga maritima. EMBO J. 19, 5300–5307. in the online version, at http://dx.doi.org/10.1016/j.febslet.2014. [20] Fujita, J., Miyazaki, Y., Hirose, M., Nagao, C., Mizohata, E., Matsumoto, Y., 04.008. Mizuguchi, K., Inoue, T. and Matsumura, H. (2013) Expression, purification, crystallization and preliminary crystallographic study of FtsA from methicillin-resistant Staphylococcus aureus. Acta Crystallogr. Sect. F Struct. References Biol. Cryst. Commun. 69, 895–898. [21] LeMaster, D.M. and Richards, F.M. 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