Cell Division Protein FtsZ Research Articles

Proteomic approach to identify host cell attachment proteins provides protective Pseudomonas aeruginosa vaccine antigen FtsZ

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that causes severe nosocomial infections in susceptible individuals due to the emergence of multidrug-resistant strains. There are no approved vaccines against P. aeruginosa infections nor candidates in active clinical development, highlighting the need for novel candidates and strategies. Using a cell-blot proteomic approach, we reproducibly identified 49 proteins involved in interactions with human lung epithelial cells across four P. aeruginosa strains. Among these were cell division protein FtsZ and outer membrane protein OpmH. Escherichia coli BL21 cells overexpressing recombinant FtsZ or rOpmH showed a 66- and 15-fold increased ability to attach to 16HBE14o− cells, further supporting their involvement in host cell attachment. Both antigens led to proliferation of NK and CD8+ cytotoxic T cells, significant increases in the production of IFN-γ, IL-17A, TNF and IL-4 in immunised mice and elicited strong antigen-specific serological IgG1 and IgG2c responses. Immunisation with FtsZ significantly reduced bacterial burden in the lungs by 1.9-log CFU and dissemination to spleen by 1.8-log CFU. The protective antigen candidate, FtsZ, would not have been identified by traditional approaches relying on either virulence mechanisms or sequence-based predictions, opening new avenues in the development of an anti-P. aeruginosa vaccine.

Staphylococcus aureus major cell division protein FtsZ assembly is inhibited by silibinin, a natural flavonolignan that also blocked bacterial growth and biofilm formation

The bacterial cell division protein FtsZ has been considered a potential therapeutic target due to its rapid treadmilling that induces cellular wall construction in bacteria. The current study discovered a novel antimicrobial compound, silibinin, a natural flavonolignan and its impact on the recombinant S. aureus FtsZ (SaFtsZ). Silibinin inhibited S. aureus Newman growth in a dose-dependent manner. The IC50 and MIC values for silibinin were 75 μM and 200 μM, respectively. It had no cytotoxicity against HEK293 cells in vitro. Silibinin also enlarged the bacterial cell morphology by ∼40 folds and showed antibiofilm property. It perturbed the S. aureus membrane potential both at IC50 conc. and at MIC conc. Further, it inhibited both the polymerization and GTPase activity of SaFtsZ. It did not inhibit tubulin assembly, a eukaryotic FtsZ homolog. A fluorescence quenching study yielded the Kd value for SaFtsZ-Silibinin interaction and binding stoichiometry 0.857 ± 0.188 μM and 1:1, respectively. Both in silico study and competition assay indicated that silibinin binds at the GTP binding site on SaFtsZ. The Ki value for the silibinin-mediated inhibition of SaFtsZ was 8.8 μM. Therefore, these findings have comprehensively shown the antimicrobial behavior of silibinin on S. aureus Newman cells targeting SaFtsZ.

Complex state transitions of the bacterial cell division protein FtsZ.

The key bacterial cell division protein FtsZ can adopt multiple conformations, and prevailing models suggest that transitions of FtsZ subunits from the closed to open state are necessary for filament formation and stability. Using all-atom molecular dynamics simulations, we analyzed state transitions of Staphylococcus aureus FtsZ as a monomer, dimer, and hexamer. We found that monomers can adopt intermediate states but preferentially adopt a closed state that is robust to forced reopening. Dimer subunits transitioned between open and closed states, and dimers with both subunits in the closed state remained highly stable, suggesting that open-state conformations are not necessary for filament formation. Mg2+ strongly stabilized the conformation of GTP-bound subunits and the dimer filament interface. Our hexamer simulations indicate that the plus end subunit preferentially closes and that other subunits can transition between states without affecting inter-subunit stability. We found that rather than being correlated with subunit opening, inter-subunit stability was strongly correlated with catalytic site interactions. By leveraging deep-learning models, we identified key intrasubunit interactions governing state transitions. Our findings suggest a greater range of possible monomer and filament states than previously considered and offer new insights into the nuanced interplay between subunit states and the critical role of nucleotide hydrolysis and Mg2+ in FtsZ filament dynamics.

Queuosine biosynthetic enzyme, QueE moonlights as a cell division regulator.

In many organisms, stress responses to adverse environments can trigger secondary functions of certain proteins by altering protein levels, localization, activity, or interaction partners. Escherichia coli cells respond to the presence of specific cationic antimicrobial peptides by strongly activating the PhoQ/PhoP two-component signaling system, which regulates genes important for growth under this stress. As part of this pathway, a biosynthetic enzyme called QueE, which catalyzes a step in the formation of queuosine (Q) tRNA modification is upregulated. When cellular QueE levels are high, it co-localizes with the central cell division protein FtsZ at the septal site, blocking division and resulting in filamentous growth. Here we show that QueE affects cell size in a dose-dependent manner. Using alanine scanning mutagenesis of amino acids in the catalytic active site, we pinpoint residues in QueE that contribute distinctly to each of its functions-Q biosynthesis or regulation of cell division, establishing QueE as a moonlighting protein. We further show that QueE orthologs from enterobacteria like Salmonella typhimurium and Klebsiella pneumoniae also cause filamentation in these organisms, but the more distant counterparts from Pseudomonas aeruginosa and Bacillus subtilis lack this ability. By comparative analysis of E. coli QueE with distant orthologs, we elucidate a unique region in this protein that is responsible for QueE's secondary function as a cell division regulator. A dual-function protein like QueE is an exception to the conventional model of "one gene, one enzyme, one function", which has divergent roles across a range of fundamental cellular processes including RNA modification and translation to cell division and stress response.

Building the Bacterial Divisome atthe Septum.

Across living organisms, division is necessary for cell survival and passing heritable information to the next generation. For this reason, cell division is highly conserved among eukaryotes and prokaryotes. Among the most highly conserved cell division proteins in eukaryotes are tubulin and actin. Tubulin polymerizes to form microtubules, which assemble into cytoskeletal structures in eukaryotes, such as the mitotic spindle that pulls chromatids apart during mitosis. Actin polymerizes to form a morphological framework for the eukaryotic cell, or cytoskeleton, that undergoes reorganization during mitosis. In prokaryotes, two of the most highly conserved cell division proteins are the tubulin homolog FtsZ and the actin homolog FtsA. In this chapter, the functions of the essential bacterial cell division proteins FtsZ and FtsA and their roles in assembly of the divisome at the septum, the site of cell division, will be discussed. In most bacteria, including Escherichia coli, the tubulin homolog FtsZ polymerizes at midcell, and this step is crucial for recruitment of many other proteins to the division site. For this reason, both FtsZ abundance and polymerization are tightly regulated by a variety of proteins. The actin-like FtsA protein polymerizes and tethers FtsZ polymers to the cytoplasmic membrane. Additionally, FtsA interacts with later stage cell division proteins, which are essential for division and for building the new cell wall at the septum. Recent studies have investigated how actin-like polymerization of FtsA on the lipid membrane may impact division, and we will discuss this and other ways that division in bacteria is regulated through FtsZ and FtsA.

ITRAQ-based quantitative proteomic analysis of the antibacterial mechanism of silver nanoparticles against multidrug-resistant Streptococcus suis.

The increase in antibiotic resistance of bacteria has become a major concern in clinical treatment. Silver nanoparticles (AgNPs) have significant antibacterial effects against Streptococcus suis. Therefore, this study aimed to investigate the antibacterial activity and mechanism of action of AgNPs against multidrug-resistant S. suis. The effect of AgNPs on the morphology of multidrug-resistant S. suis was observed using scanning electron microscopy (SEM). Differentially expressed proteins were analyzed by iTRAQ quantitative proteomics, and the production of reactive oxygen species (ROS) was assayed by H2DCF-DA staining. SEM showed that AgNPs disrupted the normal morphology of multidrug-resistant S. suis and the integrity of the biofilm structure. Quantitative proteomic analysis revealed that a large number of cell wall synthesis-related proteins, such as penicillin-binding protein and some cell cycle proteins, such as the cell division protein FtsZ and chromosomal replication initiator protein DnaA, were downregulated after treatment with 25 μg/mL AgNPs. Significant changes were also observed in the expression of the antioxidant enzymes glutathione reductase, alkyl hydroperoxides-like protein, α/β superfamily hydrolases/acyltransferases, and glutathione disulfide reductases. ROS production in S. suis positively correlated with AgNP concentration. The potential antibacterial mechanism of AgNPs may involve disrupting the normal morphology of bacteria by inhibiting the synthesis of cell wall peptidoglycans and inhibiting the growth of bacteria by inhibiting the cell division protein FtsZ and Chromosomal replication initiator protein DnaA. High oxidative stress may be a significant cause of bacterial death. The potential mechanism by which AgNPs inhibit S. suis biofilm formation may involve affecting bacterial adhesion and interfering with the quorum sensing system.

Structure-Activity Relationship Target Prediction Studies of Clindamycin Derivatives with Broad-Spectrum Bacteriostatic Antibacterial Properties.

This study investigated the potential of clindamycin derivatives with broad-spectrum antibacterial properties. The main goal was to identify new antibacterial targets to lay the foundation for developing novel antimicrobial agents. This research used molecular docking and dynamics simulations to explore how clindamycin derivatives could combat bacterial resistance and widen their antibacterial capabilities. Three different clindamycin derivatives were studied against 300 target proteins. Among these, 26 proteins were found to be common targets for all three derivatives. After further screening through molecular docking and dynamics simulations, four specific protein targets were identified. Notably, one of these targets, cell division protein FtsZ, was found to be primarily located in the cyto and cyto_nucl compartments. These findings suggest that clindamycin derivatives have the potential to address bacterial resistance and broaden their antibacterial effectiveness through these identified protein targets.

Inhibition of host cell division by T5 protein 008 (Hdi).

Bacteriophages (phages) have evolved effective methods of exploiting host-cell machinery by redirecting molecular pathways. In this study, we describe an inhibitor of bacterial growth, T5.008, encoded by the T5 phage. High-throughput sequencing of genomic DNA from bacterial mutants resistant to T5.008 identified mutations in the Escherichia coli ftsZ gene, suggesting that T5.008 targets the cell division protein FtsZ. We validated this finding by replacing a residue in FtsZ with one that confers protection from T5.008 inhibition. We used time-lapse microscopy to show that T5.008 interferes with FtsZ ring formation and stability, thereby inhibiting division. We also demonstrated that phages expressing T5.008 can prevent host division before lysis much more efficiently than those lacking T5.008. We also showed that T5.008 provides a competitive advantage to the T5 phage. This strategy of inhibiting division during phage infection likely enables T5 phage to utilize host-cell resources efficiently.IMPORTANCEWe have identified a novel phage-encoded inhibitor of the major cytoskeletal protein in bacterial division, FtsZ. The inhibition is shown to confer T5 bacteriophage with a growth advantage in dividing hosts. Our studies demonstrate a strategy in bacteriophages to maximize their progeny number by inhibiting escape of one of the daughter cells of an infected bacterium. They further emphasize that FtsZ is a natural target for bacterial growth inhibition.

Site-specific bioorthogonal protein labelling by tetrazine ligation using endogenous β-amino acid dienophiles.

The tetrazine ligation is an inverse electron-demand Diels-Alder reaction widely used for bioorthogonal modifications due to its versatility, site specificity and fast reaction kinetics. A major limitation has been the incorporation of dienophiles in biomolecules and organisms, which relies on externally added reagents. Available methods require the incorporation of tetrazine-reactive groups by enzyme-mediated ligations or unnatural amino acid incorporation. Here we report a tetrazine ligation strategy, termed TyrEx (tyramine excision) cycloaddition, permitting autonomous dienophile generation in bacteria. It utilizes a unique aminopyruvate unit introduced by post-translational protein splicing at a short tag. Tetrazine conjugation occurs rapidly with a rate constant of 0.625 (15) M-1 s-1 and was applied to produce a radiolabel chelator-modified Her2-binding Affibody and intracellular, fluorescently labelled cell division protein FtsZ. We anticipate the labelling strategy to be useful for intracellular studies of proteins, as a stable conjugation method for protein therapeutics, as well as other applications.

The C-terminal domain of MinC, a cell division regulation protein, is sufficient to form a copolymer with MinD.

Assembly of cell division protein FtsZ into the Z-ring at the division site is a key step in bacterial cell division. The Min proteins can restrict the Z-ring to the middle of the cell. MinC is the main protein that obstructs Z-ring formation by inhibiting FtsZ assembly. Its N-terminal domain (MinCN ) regulates the localization of the Z-ring by inhibiting FtsZ polymerization, while its C-terminal domain (MinCC ) binds to MinD as well as to FtsZ. Previous studies have shown that MinC and MinD form copolymers in vitro. This copolymer may greatly enhance the binding of MinC to FtsZ, and/or prevent FtsZ filaments from diffusing to the ends of the cell. Here, we investigated the assembly properties of MinCC -MinD of Pseudomonas aeruginosa. We found that MinCC is sufficient to form the copolymers. Although MinCC -MinD assembles into larger bundles, most likely because MinCC is spatially more readily bound to MinD, its copolymerization has similar dynamic properties: the concentration of MinD dominates their copolymerization. The critical concentration of MinD is around 3 μm and when MinD concentration is high enough, a low concentration MinCC could still be copolymerized. We also found that MinCC -MinD can still rapidly bind to FtsZ protofilaments, providing direct evidence that MinCC also interacts directly with FtsZ. However, although the presence of minCC can slightly improve the division defect of minC-knockout strains and shorten the cell length from an average of 12.2 ± 6.7 to 6.6 ± 3.6 μm, it is still insufficient for the normal growth and division of bacteria.

Inhibition of peptidoglycan synthesis is sufficient for total arrest of staphylococcal cell division

Bacterial cell wall biosynthesis is the target of many important antibiotics. Its spatiotemporal organization is closely coordinated with cell division. However, the role of peptidoglycan synthesis within cell division is not fully understood. Even less is known about the impact of antibiotics on the coordination of these two essential processes. Visualizing the essential cell division protein FtsZ and other key proteins in Staphylococcus aureus, we show that antibiotics targeting peptidoglycan synthesis arrest cell division within minutes of treatment. The glycopeptides vancomycin and telavancin completely inhibit septum constriction in all phases of cell division. The beta-lactam oxacillin stops division progress by preventing recruitment of the major peptidoglycan synthase PBP2 to the septum, revealing PBP2 as crucial for septum closure. Our work identifies cell division as key cellular target of these antibiotics and provides evidence that peptidoglycan synthesis is the essential driving force of septum constriction throughout cell division of S. aureus.

Homology modeling, virtual screening, molecular docking, and dynamics studies for discovering Staphylococcus epidermidis FtsZ inhibitors.

Staphylococcus epidermidis is the most common cause of medical device-associated infections and is an opportunistic biofilm former. Among hospitalized patients, S. epidermidis infections are the most prevalent, and resistant to most antibiotics. In order to overcome this resistance, it is imperative to treat the infection at a cellular level. The present study aims to identify inhibitors of the prokaryotic cell division protein FtsZ a widely conserved component of bacterial cytokinesis. Two substrate binding sites are present on the FtsZ protein; the nucleotide-binding domain and the inter-domain binding sites. Molecular modeling was used to identify potential inhibitors against the binding sites of the FtsZ protein. One hundred thirty-eight chemical entities were virtually screened for the binding sites and revealed ten molecules, each with good binding affinities (docking score range -9.549 to -4.290kcal/mol) compared to the reference control drug, i.e., Dacomitinib (-4.450kcal/mol) and PC190723 (-4.694kcal/mol) at nucleotide and inter-domain binding sites respectively. These top 10 hits were further analyzed for their ADMET properties and molecular dynamics simulations. The Chloro-derivative of GTP, naphthalene-1,3-diyl bis(3,4,5-trihydroxybenzoate), Guanosine triphosphate (GTP), morpholine and methylpiperazine derivative of GTP were identified as the lead molecules for nucleotide binding site whereas for inter-domain binding site, 1-(((amino(iminio)methyl)amino)methyl)-3-(3-(tert-butyl)phenyl)-6,7-dimethoxyisoquinolin-2-ium, and Chlorogenic acidwere identified as lead molecules. Molecular dynamics simulation and post MM/GBSA analysis of the complexes revealed good protein-ligand stability predicting them as potential inhibitors of FtsZ (Figure 1). Thus, identified FtsZ inhibitors are a promising lead compounds for S. epidermidis related infections.

Reconstitution and expression of mcy gene cluster in the model cyanobacterium Synechococcus 7942 reveals a role of MC-LR in cell division.

Cyanobacterial blooms pose a serious threat to public health due to the presence of cyanotoxins. Microcystin-LR (MC-LR) produced by Microcystis aeruginosa is the most common cyanotoxins. Due to the limitation of isolation, purification, and genetic manipulation techniques, it is difficult to study and verify insitu the biosynthetic pathways and molecular mechanisms of MC-LR. We reassembled the biosynthetic gene cluster (mcy cluster) of MC-LR invitro by synthetic biology, designed and constructed the strong bidirectional promoter biPpsbA2 , transformed it into Synechococcus 7942, and successfully expressed MC-LR at a level of 0.006-0.018 fg cell-1 d-1 . We found the expression of MC-LR led to abnormal cell division and cellular filamentation, further using various methods proved that by irreversibly competing its GTP-binding site, MC-LR inhibits assembly of the cell division protein FtsZ. The study represents the first reconstitution and expression of the mcy cluster and the autotrophic production of MC-LR in model cyanobacterium, which lays the foundation for resolving the microcystins biosynthesis pathway. The discovered role of MC-LR in cell division reveals a mechanism of how blooming cyanobacteria gain a competitive edge over their nonblooming counterparts.

A mechanism of salt bridge-mediated resistance to FtsZ inhibitor PC190723 revealed by a cell-based screen.

Bacterial cell division proteins, especially the tubulin homologue FtsZ, have emerged as strong targets for developing new antibiotics. Here, we have utilized the fission yeast heterologous expression system to develop a cell-based assay to screen for small molecules that directly and specifically target the bacterial cell division protein FtsZ. The strategy also allows for simultaneous assessment of the toxicity of the drugs to eukaryotic yeast cells. As a proof-of-concept of the utility of this assay, we demonstrate the effect of the inhibitors sanguinarine, berberine, and PC190723 on FtsZ. Though sanguinarine and berberine affect FtsZ polymerization, they exert a toxic effect on the cells. Further, using this assay system, we show that PC190723 affects Helicobacter pylori FtsZ function and gain new insights into the molecular determinants of resistance to PC190723. On the basis of sequence and structural analysis and site-specific mutations, we demonstrate that the presence of salt bridge interactions between the central H7 helix and β-strands S9 and S10 mediates resistance to PC190723 in FtsZ. The single-step in vivo cell-based assay using fission yeast enabled us to dissect the contribution of sequence-specific features of FtsZ and cell permeability effects associated with bacterial cell envelopes. Thus, our assay serves as a potent tool to rapidly identify novel compounds targeting polymeric bacterial cytoskeletal proteins like FtsZ to understand how they alter polymerization dynamics and address resistance determinants in targets.

Gossypol acetate: A natural polyphenol derivative with antimicrobial activities against the essential cell division protein FtsZ.

Antimicrobial resistance has attracted worldwide attention and remains an urgent issue to resolve. Discovery of novel compounds is regarded as one way to circumvent the development of resistance and increase the available treatment options. Gossypol is a natural polyphenolic aldehyde, and it has attracted increasing attention as a possible antibacterial drug. In this paper, we studied the antimicrobial properties (minimum inhibitory concentrations) of gossypol acetate against both Gram-positive and Gram-negative bacteria strains and dig up targets of gossypol acetate using in vitro assays, including studying its effects on functions (GTPase activity and polymerization) of Filamenting temperature sensitive mutant Z (FtsZ) and its interactions with FtsZ using isothermal titration calorimetry (ITC), and in vivo assays, including visualization of cell morphologies and proteins localizations using a microscope. Lastly, Bacterial membrane permeability changes were studied, and the cytotoxicity of gossypol acetate was determined. We also estimated the interactions of gossypol acetate with the promising target. We found that gossypol acetate can inhibit the growth of Gram-positive bacteria such as the model organism Bacillus subtilis and the pathogen Staphylococcus aureus [both methicillin-sensitive (MSSA) and methicillin-resistant (MRSA)]. In addition, gossypol acetate can also inhibit the growth of Gram-negative bacteria when the outer membrane is permeabilized by Polymyxin B nonapeptide (PMBN). Using a cell biological approach, we show that gossypol acetate affects cell division in bacteria by interfering with the assembly of the cell division FtsZ ring. Biochemical analysis shows that the GTPase activity of FtsZ was inhibited and polymerization of FtsZ was enhanced in vitro, consistent with the block to cell division in the bacteria tested. The binding mode of gossypol acetate in FtsZ was modeled using molecular docking and provides an understanding of the compound mode of action. The results point to gossypol (S2303) as a promising antimicrobial compound that inhibits cell division by affecting FtsZ polymerization and has potential to be developed into an effective antimicrobial drug by chemical modification to minimize its cytotoxic effects in eukaryotic cells that were identified in this work.

A secreted effector with a dual role as a toxin and as a transcriptional factor

Bacteria have evolved multiple secretion systems for delivering effector proteins into the cytosol of neighboring cells, but the roles of many of these effectors remain unknown. Here, we show that Yersinia pseudotuberculosis secretes an effector, CccR, that can act both as a toxin and as a transcriptional factor. The effector is secreted by a type VI secretion system (T6SS) and can enter nearby cells of the same species and other species (such as Escherichia coli) via cell-cell contact and in a contact-independent manner. CccR contains an N-terminal FIC domain and a C-terminal DNA-binding domain. In Y. pseudotuberculosis cells, CccR inhibits its own expression by binding through its DNA-binding domain to the cccR promoter, and affects the expression of other genes through unclear mechanisms. In E. coli cells, the FIC domain of CccR AMPylates the cell division protein FtsZ, inducing cell filamentation and growth arrest. Thus, our results indicate that CccR has a dual role, modulating gene expression in neighboring cells of the same species, and inhibiting the growth of competitors.

Lipid Surfaces and Glutamate Anions Enhance Formation of Dynamic Biomolecular Condensates Containing Bacterial Cell Division Protein FtsZ and Its DNA-Bound Regulator SlmA.

Dynamic biomolecular condensates formed by liquid-liquid phase separation can regulate the spatial and temporal organization of proteins, thus modulating their functional activity in cells. Previous studies showed that the cell division protein FtsZ from Escherichia coli formed dynamic phase-separated condensates with nucleoprotein complexes containing the FtsZ spatial regulator SlmA under crowding conditions, with potential implications for condensate-mediated spatiotemporal control of FtsZ activity in cell division. In the present study, we assessed formation of these condensates in the presence of lipid surfaces and glutamate ions to better approximate the E. coli intracellular environment. We found that potassium glutamate substantially promoted the formation of FtsZ-containing condensates when compared to potassium chloride in crowded solutions. These condensates accumulated on supported lipid bilayers and eventually fused, resulting in a time-dependent increase in the droplet size. Moreover, the accumulated condensates were dynamic, capturing protein from the external phase. FtsZ partitioned into the condensates at the lipid surface only in its guanosine diphosphate (GDP) form, regardless of whether it came from FtsZ polymer disassembly upon guanosine triphosphate (GTP) exhaustion. These results provide insights into the behavior of these GTP-responsive condensates in minimal membrane systems, which suggest how these membraneless assemblies may tune critical bacterial division events during the cell cycle.

Connecting sequence features within the disordered C-terminal linker of Bacillus subtilis FtsZ to functions and bacterial cell division

Intrinsically disordered regions (IDRs) can function as autoregulators of folded enzymes to which they are tethered. One example is the bacterial cell division protein FtsZ. This includes a folded core and a C-terminal tail (CTT) that encompasses a poorly conserved, disordered C-terminal linker (CTL) and a well-conserved 17-residue C-terminal peptide (CT17). Sites for GTPase activity of FtsZs are formed at the interface between GTP binding sites and T7 loops on cores of adjacent subunits within dimers. Here, we explore the basis of autoregulatory functions of the CTT in Bacillus subtilis FtsZ (Bs-FtsZ). Molecular simulations show that the CT17 of Bs-FtsZ makes statistically significant CTL-mediated contacts with the T7 loop. Statistical coupling analysis of more than 1,000 sequences from FtsZ orthologs reveals clear covariation of the T7 loop and the CT17 with most of the core domain, whereas the CTL is under independent selection. Despite this, we discover the conservation of nonrandom sequence patterns within CTLs across orthologs. To test how the nonrandom patterns of CTLs mediate CTT-core interactions and modulate FtsZ functionalities, we designed Bs-FtsZ variants by altering the patterning of oppositely charged residues within the CTL. Such alterations disrupt the core-CTT interactions, lead to anomalous assembly and inefficient GTP hydrolysis invitro and protein degradation, aberrant assembly, and disruption of cell division invivo. Our findings suggest that viable CTLs in FtsZs are likely to be IDRs that encompass nonrandom, functionally relevant sequence patterns that also preserve three-way covariation of the CT17, the T7 loop, and core domain.

Halogenation of tyrosine perturbs large-scale protein self-organization

Protein halogenation is a common non-enzymatic post-translational modification contributing to aging, oxidative stress-related diseases and cancer. Here, we report a genetically encodable halogenation of tyrosine residues in a reconstituted prokaryotic filamentous cell-division protein (FtsZ) as a platform to elucidate the implications of halogenation that can be extrapolated to living systems of much higher complexity. We show how single halogenations can fine-tune protein structures and dynamics of FtsZ with subtle perturbations collectively amplified by the process of FtsZ self-organization. Based on experiments and theories, we have gained valuable insights into the mechanism of halogen influence. The bending of FtsZ structures occurs by affecting surface charges and internal domain distances and is reflected in the decline of GTPase activities by reducing GTP binding energy during polymerization. Our results point to a better understanding of the physiological and pathological effects of protein halogenation and may contribute to the development of potential diagnostic tools.

Discovery of Epipodophyllotoxin-Derived B2 as Promising XooFtsZ Inhibitor for Controlling Bacterial Cell Division: Structure-Based Virtual Screening, Synthesis, and SAR Study.

The emergence of phytopathogenic bacteria resistant to antibacterial agents has rendered previously manageable plant diseases intractable, highlighting the need for safe and environmentally responsible agrochemicals. Inhibition of bacterial cell division by targeting bacterial cell division protein FtsZ has been proposed as a promising strategy for developing novel antibacterial agents. We previously identified 4′-demethylepipodophyllotoxin (DMEP), a naturally occurring substance isolated from the barberry species Dysosma versipellis, as a novel chemical scaffold for the development of inhibitors of FtsZ from the rice blight pathogen Xanthomonas oryzae pv. oryzae (Xoo). Therefore, constructing structure−activity relationship (SAR) studies of DMEP is indispensable for new agrochemical discovery. In this study, we performed a structure−activity relationship (SAR) study of DMEP derivatives as potential XooFtsZ inhibitors through introducing the structure-based virtual screening (SBVS) approach and various biochemical methods. Notably, prepared compound B2, a 4′-acyloxy DMEP analog, had a 50% inhibitory concentration of 159.4 µM for inhibition of recombinant XooFtsZ GTPase, which was lower than that of the parent DMEP (278.0 µM). Compound B2 potently inhibited Xoo growth in vitro (minimum inhibitory concentration 153 mg L−1) and had 54.9% and 48.4% curative and protective control efficiencies against rice blight in vivo. Moreover, compound B2 also showed low toxicity for non-target organisms, including rice plant and mammalian cell. Given these interesting results, we provide a novel strategy to discover and optimize promising bactericidal compounds for the management of plant bacterial diseases.