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Both the excitation and emission wavelengths were set at 350 nm and a slit width of 4 nm was used.The hydrolysis reaction was initiated by addition of 5 mM MgCl 2 and 1 mM GTP.Molecular docking was employed to identify the most favorable position of the respective ligands, namely GDP, GTP, (S)-rhodomyrtone, and (R)-rhodomyrtone, upon interactions with FtsZ.At various time pionts after treatment, samples were placed onto 1% agarose slides and images were taken using a Nikon Eclipse Ti microscope as specified above.Whois Server Version 2.1.3 Domain: CHULA.AC.TH Registrar: T.H.NIC Co., Ltd. Name Server:.The total binding free energy of the ligands (rhodomyrtone and GDP) as well as their energy contribution determined and were used to assess the binding affinity of the ligand to FtsZ.In fact, fractionation experiments with S. aureus showed that rhodomyrtone is unable to cross the cytoplasmic membrane barrier to reach the cytosol in the first 4 h of treatment but instead accumulates in the cell debris ( Data S3 ).Visutthi M, Srimanote P, Voravuthikunchai SP. 2011. Responses in the expression of extracellular proteins in methicillin-resistant Staphylococcus aureus treated with rhodomyrtone.

Therefore, mid-cell localization of FtsZ is crucial for the cell division process.Docking studies were carried out using the Autodock4 package ( Morris et al., 2009 ) to predict the most convenient conformation and ligand position bound to the protein.This approach is able to provide information at atomic levels by calculating the interactions between ligands and receptors and predicting conformational changes in drug-binding targets.Loop H6 and loop H7 are important for binding one FtsZ monomer to another to finally form FtsZ filaments ( Matsui et al., 2014 ). Therefore, conformational changes in this region may reflect FtsZ polymerization.Kollman PA, Massova I, Reyes C, Kuhn B, Huo S, Chong L, Lee M, Lee T, Duan Y, Wang W. 2000. Calculating structures and free energies of complex molecules: combining molecular mechanics and continuum models.

Conditional mutants defective in cell division elongate into filaments.The free energy of each state was derived from molecular mechanics energy, broken down into.

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Parameter files, library files, and the last snapshot of all FtsZ states.

To get insights into the molecular interactions of rhodomyrtone with FtsZ, we compared the structures of FtsZ bound to rhodomyrtone, in both its (R)- and (S)-enantiomeric forms ( Fig. 1 ), with the natural states of FtsZ, namely the nucleotide-free and two nucleotide-bound forms (GDP and GTP).Electrostatic forces significantly contributed to the binding of GDP to FtsZ as evidenced by the strong interaction between the beta-phosphate of GDP and arginine (Arg135) at the binding site.Pilhofer M, Ladinsky MS, McDowall AW, Petroni G, Jensen GJ. 2011. Microtubules in bacteria: ancient tubulins build a five-protofilament homolog of the eukaryotic cytoskeleton.The following information was supplied regarding data availability.Smaller rmsf values in the GTP-binding state imply less flexibility of the protein structure.The sample was loaded onto a 5 mL HiTrap Q HP column (GE Healthcare) equilibrated with 3 column volumes of buffer A.Localization of FtsZ was not affected by inhibitory rhodomyrtone concentrations, showing that FtsZ is not the main in vivo target.

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Mukherjee S, Robinson CA, Howe AG, Mazor T, Wood PA, Urgaonkar S, Hebert AM, RayChaudhuri D, Shaw JT. 2007. N-Benzyl-3-sulfonamidopyrrolidines as novel inhibitors of cell division in Escherichia coli.We further investigated the effects of rhodomyrtone on FtsZ in vitro and in vivo.

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Additionally, in the (S)-rhodomyrtone state, we observed differences from the GDP- and GTP-binding states in loop H6 and loop H7.The column was washed with buffer A until reaching a stable baseline, followed by washing with 5% buffer B (50 mM MES-KOH pH 6.5, 5 mM MgCl 2, 1 M KCl).Instead, it is likely that rhodomyrtone interacts with membrane or cell wall-bound protein targets or with membrane or cell wall structural components, and delocalization of divisome proteins is a result of this interaction.

This work was supported by Higher Education Research Promotion and National Research University Project of Thailand, Office of Higher Education Commission and TRF Senior Research Scholar grant (Grant No.Czaplewski LG, Collins I, Boyd EA, Brown D, East SP, Gardiner M, Fletcher R, Haydon DJ, Henstock V, Ingram P. 2009. Antibacterial alkoxybenzamide inhibitors of the essential bacterial cell division protein FtsZ.In this study, we investigated the structure of FtsZ bound to various ligands as well as ligand-free FtsZ using molecular dynamics simulation.

Rhodomyrtone, a natural compound containing (S)-rhodomyrtone and (R)-rhodomyrtone, reduced FtsZ assembly in a concentration-dependent manner by maximally 36%.A docked complex structure was chosen on the basis of the lowest binding energy.Strahl H, Hamoen LW. 2010. Membrane potential is important for bacterial cell division.Fluorescence light microscopy finally gave insights into the effect of rhodomyrtone on FtsZ in live bacteria.

In fact, in vitro experiments with purified FtsZ revealed that both polymerization and GTPase activity were affected by rhodomyrtone.Our findings suggest an enantiomeric specificity of rhodomyrtone towards the FtsZ protein.

Glycerol was added to a final concentration of 10% prior to flash freezing.After 500 ps, the restraint on the protein and ligand components was released and the system was switched into an isobaric-isothermal (NPT) simulation at a constant pressure of 1 atm, and 310 K with a time step of 2 fs for 150 ns.LWH was funded by the Netherlands Organization for Scientific Research (NWO,, STW-Vici 12128).After reaching a stable baseline again, FtsZ was eluted in a gradient up to 50% buffer B over 5 column volumes.

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Using polymerization (light scattering) and GTPase activity assays, we found that rhodomyrtone affected the function of purified FtsZ.Ohashi Y, Chijiiwa Y, Suzuki K, Takahashi K, Nanamiya H, Sato T, Hosoya Y, Ochi K, Kawamura F. 1999. The lethal effect of a benzamide derivative, 3-methoxybenzamide, can be suppressed by mutations within a cell division gene, ftsZ, in Bacillus subtilis.