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Probing FtsZ and Tubulin with C8-Substituted GTP Analogs Reveals Differences in Their Nucleotide Binding Sites
Abstract
The cytoskeletal proteins, FtsZ and tubulin, play a pivotal role in prokaryotic cell division and eukaryotic chromosome segregation, respectively. Selective inhibitors of the GTP-dependent polymerization of FtsZ could constitute a new class of antibiotics, while several inhibitors of tubulin are widely used in antiproliferative therapy. In this work, we set out to identify selective inhibitors of FtsZ based on the structure of its natural ligand, GTP. We found that GTP analogs with small hydrophobic substituents at C8 of the nucleobase efficiently inhibit FtsZ polymerization, whereas they have an opposite effect on the polymerization of tubulin. The inhibitory activity of the GTP analogs on FtsZ polymerization allowed us to crystallize FtsZ in complex with C8-morpholino-GTP, revealing the binding mode of a GTP derivative containing a nonmodified triphosphate chain.
... Whereas tubulin is a classical target of numerous anticancer drugs, discovering new useful antibiotics targeting FtsZ remains a challenge. Over one hundred X-ray structures of tubulin-inhibitor complexes are available [9], but only a handful of FtsZ small-molecule complex structures have been reported to date [10][11][12][13][14][15][16], suggesting a lesser degree of druggability of FtsZ compared to tubulin. Nevertheless, considerable knowledge has been acquired about the structure, assembly dynamics, and function of FtsZ filaments and the Z-ring, the FtsZ partner proteins, and several small molecule chemotypes that specifically interact with FtsZ. ...
... The bound GDP nucleotide, a co-solvent molecule and one benzamide allosteric inhibitor are shown (sticks colored by atom type) [PDB 6YD6, [16]]. (D) AaFtsZ monomer in complex with 8-morpholino-GTP [PDB 2r7l, [10]]. (E) MjFtsZ monomer in complex with 8-pyrrolidino-GTP; NMR-based ligand model after docking and MD of the protein complex (adapted with permission from [48]; copyright 2013 American Chemical Society). ...
... However, both proteins share limited sequence identity, and it was soon realized that typical tubulin inhibitors do not inhibit FtsZ, whereas FtsZ inhibitors infrequently act on tubulin. A difference between the interfacial active sites of FtsZ and tubulin was demonstrated with GTP analogs bearing small hydrophobic substituents at the C8 position of the guanine ring, such as 8-methoxy-GTP and 8-Br-GTP, which bind with high affinity and selectively inhibit FtsZ polymerization in vitro, while promoting microtubule assembly and being substrates for tubulin GTPase [10]. The FtsZ assembly inhibitory activity of C8-GTP derivatives correlates with their binding affinity [10,48]. ...
The global spread of bacterial antimicrobial resistance is associated to millions of deaths from bacterial infections per year, many of which were previously treatable. This, combined with slow antibiotic deployment, has created an urgent need for developing new antibiotics. A still clinically unexploited mode of action consists in suppressing bacterial cell division. FtsZ, an assembling GTPase, is the key protein organizing division in most bacteria and an attractive target for antibiotic discovery. Nevertheless, developing effective antibacterial inhibitors targeting FtsZ has proven challenging. Here we review our decade-long multidisciplinary research on small molecule inhibitors of bacterial division, in the context of global efforts to discover FtsZ-targeting antibiotics. We focus on methods to characterize synthetic inhibitors that either replace bound GTP from the FtsZ nucleotide binding pocket conserved across diverse bacteria or selectively bind into the allosteric site at the interdomain cleft of FtsZ from Bacillus subtilis and the pathogen Staphylococcus aureus. These approaches include phenotype screening combined with fluorescence polarization screens for ligands binding into each site, followed by detailed cytological profiling, and biochemical and structural studies. The results are analyzed to design an optimized workflow to identify effective FtsZ inhibitors, and new approaches for the discovery of FtsZ-targeting antibiotics are discussed.
... An inhibitor bound to the cleft will prevent the T7 loop involvement in GTP hydrolysis thereby losing energy supply for FtsZ polymerization [7,[25][26][27][28][29]. Many synthetic and natural compounds have been screened to target the GTP binding site of FtsZ [30][31][32][33][34][35]. Many secondary metabolites of plants are known to serve as antimicrobial agents and many spices consumed by humans also have medicinal values. ...
... In this study, we observed that the recombinantly purified stFtsZ form unstable oligomers and upon GTP binding it forms stable oligomers shown by DLS and gel filtration experiments. Many synthetic and natural compounds have been screened to target the GTP binding site of FtsZ to inhibit its polymerization [16,23,[30][31][32][33][34][35][44][45][46]. To disrupt the polymerization process we used natural compounds Eugenol, Cinnamaldehyde, Scopoletin, Quercetin and Berberine chloride. ...
Salmonella Typhi is emerging as a drug-resistant pathogen, particularly in developing countries. Hence, the progressive development of new antibiotics against novel drug targets is essential to prevent the spread of infections and mortality. The cell division protein FtsZ is an ideal drug target as the cell wall synthesis in bacteria is driven by the dynamic treadmilling nature of the FtsZ. The polymerization of the FtsZ provides the essential mechanical constricting force and flexibility to modulate the cell wall synthesis. Any alteration in FtsZ polymerization leads to the bactericidal or bacteriostatic effect. In this study, we have evaluated the secondary metabolites of natural compounds berberine chloride, cinnamaldehyde, scopoletin, quercetin and eugenol as potential inhibitors of FtsZ from Salmonella Typhi (stFtsZ) using computational, biochemical, and in vivo cell-based assays. Out of these five compounds, berberine chloride and cinnamaldehyde exhibited the best binding affinity of Kd = 7 μM and 10 μM, respectively and inhibit stFtsZ GTPase activity and polymerization by 70 %. The compound berberine chloride showed the best MIC of 500 μg/mL and 175 μg/mL against gram-negative and gram-positive bacterial strains. The findings support that these natural compounds can be used as a backbone structure to develop a broad spectrum of antibacterial agents.
... On searching for FtsZ inhibitors, detection of FtsZ polymerization either in vivo or in vitro is available; crystal structures of FtsZ from several bacterial species are available [22][23][24][25]; FtsZ polymerization state can be in vitro assayed by light scattering [26] or colorimetry [27]; the localization of FtsZ in cell can be monitored by FtsZfluorescent protein fusions [28]; details of the formed filaments can be examined by electron microscopy [29]. ...
... Thus, blocking the GTP-binding site in FtsZ reduces the available FtsZ molecules under critical concentration for polymerization [30]; and it also prevents GTPase activity to dissociate FtsZ polymer [31]. Most of these inhibitors are GTP analogs with bulky substitutions at the C8 position [22,32,33]. The GTP analogs bind FtsZ, but their bulky substitutions prevent the association of a second FtsZ subunit and consequently inhibit the GTPase activity due to the absence of the second molecule. ...
Antimicrobial targets should be essential to the life or pathogenicity of bacteria and contain conserved target binding regions. This chapter reviews the antimicrobial target sites, their structures, roles, and their inhibition. Life-essential targets include FtsZ and their regulatory proteins; they mediate the cell division and its accompanying modifications in the cell wall. Peptidoglycan biosynthesis enzymes also belong to life-essential targets; we focused on the integral membrane protein MraY, the membrane-associated protein MurG, and penicillin-binding proteins (PBPs) rather than Mur enzymes due to their in vivo inaccessibility. Targeting DNA, as an essential element, may damage the strands or interfere with the replication mechanisms, or even specific genes; sequence-specific binders are designed. For expanding the drug targets, bacterial quorum sensing systems (QSs) are targeted; it regulates several genes; we reviewed the quorum quenching through several approaches. Other targets would provide new anti-virulence drugs. The d-alanylation of teichoic acid represents a potential target in Gram-positive bacteria; we discussed the specificity and inter-species conservation of the key enzyme (d-Alanyl carrier protein ligase) in this pathway.
... In this context, a huge study was that performed by Läppchen and Andreu and their research groups. They started designing and synthetizing a structurally diverse series of 8substituted GTP analogues [58][59][60] and investigated their effect on both FtsZ and tubulin, calculated their ability to interfere with FtsZ polymerization and GTPase activity, and evaluated the antimicrobial activity of their prodrugs. ...
... The former was the first to be discovered [59]; it was proven to inhibit both E. coli FtsZ polymerization (IC50 = 37 μM) and its associated GTPase activity (IC50 = 60.2 μM). N20, designed a few years later [60], was even more potent, with an IC50 on FtsZ polymerization of 10 μM and a similar IC50 on GTPase activity (15 μM). ...
Binary fission is the most common mode of bacterial cell division and is mediated by a multiprotein complex denominated the divisome. The constriction of the Z-ring splits the mother bacterial cell into two daughter cells of the same size. The Z-ring is formed by the polymerization of FtsZ, a bacterial protein homologue of eukaryotic tubulin, and it represents the first step of bacterial cytokinesis. The high grade of conservation of FtsZ in most prokaryotic organisms and its relevance in orchestrating the whole division system make this protein a fascinating target in antibiotic research. Indeed, FtsZ inhibition results in the complete blockage of the division system and, consequently, in a bacteriostatic or a bactericidal effect. Since many papers and reviews already discussed the physiology of FtsZ and its auxiliary proteins, as well as the molecular mechanisms in which they are involved, here, we focus on the discussion of the most compelling FtsZ inhibitors, classified by their main protein binding sites and following a medicinal chemistry approach.
... To block the GTP-dependent polymerisation of FtsZ, C8-substituted GTP analogues have been tested and were shown to inhibit FtsZ polymerisation in vitro 149,150 . The small substitutions did not inhibit tubulin assembly 150 . ...
... To block the GTP-dependent polymerisation of FtsZ, C8-substituted GTP analogues have been tested and were shown to inhibit FtsZ polymerisation in vitro 149,150 . The small substitutions did not inhibit tubulin assembly 150 . Unfortunately, the C8-substituted guanine, guanosine, guanosine-5′monophosphate (GMP), and guanosine-5′-triphosphates were later found to be ineffective antibacterial agents against E. coli, despite the strong inhibition of FtsZ polymerisation in vitro; moreover, the lack of toxicity was not due to low intracellular accumulation, nor to the absence of conversion of the analogue to the inhibitory triphosphate form 151 . ...
Antibiotics save many lives, but their efficacy is under threat: overprescription, populationgrowth, and global travel all contribute to the rapid origination and spread of resistantstrains. Exacerbating this threat is the fact that no new major classes of antibioticshave been discovered in the last 30 years: this is the “discovery void.” We discuss thetraditional molecular targets of antibiotics as well as putative novel targets.
... 25 In this context, we employed mant-GTP to probe the kinetics and energetics of FtsZ-nucleotide interactions 26 and established a competitive assay using the fluorescence polarization of bound mant-GTP to determine inhibitor binding to the FtsZ nucleotide-binding site. 27 We have also employed this mant fluorophore-based assay to investigate the mechanism of C-8 substituted nucleotide FtsZ inhibitors 28,29 and to identify new GTP-replacing inhibitors of bacterial cell division that are active on resistant pathogens. 30−32 Remarkably, the 2′/3′-mant-GTP isomer mixture behaved as a single ligand species in these studies. ...
... Interestingly, the aromatic mant group disables a π-stacking interaction between conserved FtsZ residues Phe 162 (loop T5) and Phe 208 (helix 7; Figure 4E−H), among other interactions made by mant-GTP ( Figure S6). The nucleotide site of FtsZ monomers thus exhibits considerable plasticity, 27 demonstrating the ability to bind synthetic inhibitors, 30,32 GTP analogs substituted at C8 in the guanine ring, 28,29 and the mant derivatives at the ribose O2′ and O3′ positions described herein. ...
FtsZ is the organizer of cell division in most bacteria and a target in the quest for new antibiotics. FtsZ is a tubulin-like GTPase, in which the active site is completed at the interface with the next subunit in an assembled FtsZ filament. Fluorescent mant-GTP has been extensively used for competitive binding studies of nucleotide analogs and synthetic GTP-replacing inhibitors possessing antibacterial activity. However, its mode of binding and whether the mant tag interferes with FtsZ assembly function were unknown. Mant-GTP exists in equilibrium as a mixture of C2'- and C3'-substituted isomers. We have unraveled the molecular recognition process of mant-GTP by FtsZ monomers. Both isomers bind in the anti glycosidic bond conformation: 2'-mant-GTP in two ribose puckering conformations and 3'-mant-GTP in the preferred C2'endo conformation. In each case, the mant tag strongly interacts with FtsZ at an extension of the GTP binding site, which is also supported by molecular dynamics simulations. Importantly, mant-GTP binding induces archaeal FtsZ polymerization into inactive curved filaments that cannot hydrolyze the nucleotide, rather than straight GTP-hydrolyzing assemblies, and also inhibits normal assembly of FtsZ from the Gram-negative bacterium Escherichia coli, but is hydrolyzed by FtsZ from Gram-positive Bacillus subtilis. Thus, the specific interactions provided by the fluorescent mant tag indicate a new way to search for synthetic FtsZ inhibitors that selectively suppress the cell division of bacterial pathogens.
... Several tridimensional structures of the protein FtsZ from various organisms, and with various ligands in the nucleotide binding pocket have been solved using X-ray crystallographic experiments. These structures include the Methanocaldococcus jannaschii FtsZ bound to a GTP, GDP or a guanosine-5′-(α,β)-methyleno-triphosphate (GMPCPP) ( Lowe & Amos, 1998;Oliva, Cordell, & Lowe, 2004;Oliva, Trambaiolo, & Lowe, 2007), the Bacillus subtilis FtsZ bound to a 5′-guanosine-diphosphatemonothiophosphate (GTPγS) or GDP ( Oliva et al., 2007;Raymond et al., 2009), the Mycobacterium tuberculosis FtsZ bound to GTPγS, GDP or a citrate anion (CIT) ( Leung et al., 2004), the Aquifex aeolicus FtsZ bound to a 8-morpholin-4-ylguanosine-5′-tetrahydrogentriphosphate (MorphGTP) or GDP ( Lappchen et al., 2008;Oliva et al., 2007), and the Thermotoga maritima FtsZ-GMPCPP ( Oliva et al., 2004) and Pseudomonas aeruginosa FtsZ-GDP complexes ( Oliva et al., 2007). The protein FtsZ structure consists in two globular domains, the N-terminal domain containing the nucleotide binding site and the C-terminal domain whose loop T7 is involved in the GTPase activity of FtsZ. ...
... (The name and numbering of the amino-acids and secondary structures in this article are those used in reference ( Oliva et al., 2007) for the M. jannaschii FtsZ, and are recalled in Figures 1 and 2). Within the same organism, it appears that the bound nucleotide chemical nature does not influence the overall protein conformation: No large "domain movements" or significant local backbone rearrangements were detected when superimposing the different crystallographic structures of the M. jannaschii ( Oliva et al., 2004Oliva et al., , 2007), the B. subtilis ( Raymond et al., 2009), or the A. aeolicus FtsZ ( Lappchen et al., 2008;Oliva et al., 2007). However, in the M. tuberculosis FtsZ dimer, the crystallographic data indicates that the presence or absence of a γ-phosphate in the binding site modulates a structural reorganization of loop T3 and a coupled α-to-β switch of helix H2 ( Leung et al., 2004). ...
We present here a structural analysis of ten extensive all-atom molecular dynamics simulations of the monomeric protein FtsZ in various binding states. Since the polymerization and GTPase activities of FtsZ depend on the nature of a bound nucleotide as well as on the presence of a magnesium ion, we studied the structural differences between the average conformations of the following five systems: FtsZ-Apo, FtsZ-GTP, FtsZ-GDP, FtsZ-GTP-Mg, and FtsZ-GDP-Mg. The in silico solvated average structure of FtsZ-Apo significantly differs from the crystallographic structure 1W59 of FtsZ which was crystallized in a dimeric form without nucleotide and magnesium. The simulated Apo form of the protein also clearly differs from the FtsZ structures when it is bound to its ligand, the most important discrepancies being located in the loops surrounding the nucleotide binding pocket. The three average structures of FtsZ-GTP, FtsZ-GDP, and FtsZ-GTP-Mg are overall similar, except for the loop T7 located at the opposite side of the binding pocket and whose conformation in FtsZ-GDP notably differs from the one in FtsZ-GTP and FtsZ-GTP-Mg. The presence of a magnesium ion in the binding pocket has no impact on the FtsZ conformation when it is bound to GTP. In contrast, when the protein is bound to GDP, the divalent cation causes a translation of the nucleotide outwards the pocket, inducing a significant conformational change of the loop H6-H7 and the top of helix H7.
... Several approaches to screen small molecules targeting bacterial cell division and FtsZ have been established. While in vitro methods such as NMR (Domadia et al. 2007;Sun et al. 2014;Araújo-Bazán et al. 2019) and crystallography (Läppchen et al. 2008;Fujita et al. 2017) are valuable and offer information on distinct binding sites, these are not efficient for screening. Electron microscopic examination can distinguish the effects of the compounds being tested on the FtsZ protofilament assembly and lateral associations (Nova et al. 2007;Kaul et al. 2012;Anderson et al. 2012;Sun et al. 2014;Huecas et al. 2017;Kumar et al. 2011;Park et al. 2014). ...
Bacterial cell division proteins, especially the tubulin homolog 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. Based on sequence and structural analysis and site-specific mutations, we demonstrate that the presence of salt-bridge interactions between the central H7 helix and beta-strands S9 and S10 mediate 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.
... The minimal system studied here 24 is a one-component biological system reconstituted in vitro from scratch with the purified FtsZ protein (the ftsZ gene product), the known prokaryotic homologue of the eukaryotic protein tubulin (Fig. 1a). The protein has been shown to be sensitive to halogenating chemicals 25 and its activity is sensitive to modifications at individual sites 26 . As an essential part of the bacterial division ring, known as "Z ring", FtsZ has shown intriguing self-organization when reconstituted in vitro on biological membranes. ...
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.
... Some of them also ingested through meat into the human body [145]. Certain studies show deleterious effects, delayed germination and post germinative development in plants due to antibiotics [131]. The presence of antibiotics in soil, groundwater, poultry meat and other plant and animal-based products and the side effects caused by antibiotics in environment are staggering which has caused the scientific community and agencies to reassess their use [146][147][148][149][150][151][152][153]. ...
Antibiotic resistance is a major emerging issue in the health care sector, as highlighted by the WHO. Filamentous Thermosensitive mutant Z (Fts-Z) is gaining significant attention in the scientific community as a potential anti-bacterial target for fighting antibiotic resistance among several pathogenic bacteria. The Fts-Z plays a key role in bacterial cell division by allowing Z ring formation. Several in vitro and in silico experiments have demonstrated that inhibition of Fts-Z can lead to filamentous growth of the cells, and finally, cell death occurs. Many natural compounds that have successfully inhibited Fts-Z are also studied. This review article intended to highlight the structural–functional aspect of Fts-Z that leads to Z-ring formation and its contribution to the biochemistry and physiology of cells. The current trend of natural inhibitors of Fts-Z protein is also covered.
... On the contrary, different MjFtsZ structures showed only negligible differences in the T3 loop region when bound to either GDP or GTP compared with apo-MjFtsZ (PDB: 1W58, 1W5B, 1W59, 1FSZ) [7,11]. Nucleotide-dependent conformational changes were also not observed for Aquifex aeolicus FtsZ (AaFtsZ) comparing crystal structures of AaFtsZ:GDP and AaFtsZ:8-morpholino-GTP [9,36]. Further, the crystal structure of SaFtsZ bound to either GTP or GDP did not show a conformational switch of the T3 loop, but revealed an opened and a closed conformation of the FtsZ core [37]. ...
Antimicrobial resistance to virtually all clinically applied antibiotic classes severely limits the available options to treat bacterial infections. Hence, there is an urgent need to develop and evaluate new antibiotics and targets with resistance-breaking properties. Bacterial cell division has emerged as a new antibiotic target pathway to counteract multidrug-resistant pathogens. New approaches in antibiotic discovery and bacterial cell biology helped to identify compounds that either directly interact with the major cell division protein FtsZ, thereby perturbing the function and dynamics of the cell division machinery, or affect the structural integrity of FtsZ by inducing its degradation. The impressive antimicrobial activities and resistance-breaking properties of certain compounds validate the inhibition of bacterial cell division as a promising strategy for antibiotic intervention.
... However, the curved or bent confirmation of a FtsZ dimer was only recently observed at the atomic level (114,139). Li et al. (114) found that a GDP-bound Mycobacterium tuberculosis FtsZ protofilament exhibits a curved conformation, in contrast to the straight conformation that was invariably observed in nearly all previous FtsZ structures from other bacterial species (108,111,139,158,180,213). Surprisingly, the GDP-bound open conformation causes the C-terminal surface of the Mtb FtsZ protofilament to bend toward but not away from the membrane (Figure 8b). ...
The FtsZ protein is a highly conserved bacterial tubulin homolog. In vivo, the functional form of FtsZ is the polymeric, ring-like structure (Z-ring) assembled at the future division site during cell division. While it is clear that the Z-ring plays an essential role in orchestrating cytokinesis, precisely what its functions are and how these functions are achieved remain elusive. In this article, we review what we have learned during the past decade about the Z-ring's structure, function, and dynamics, with a particular focus on insights generated by recent high-resolution imaging and single-molecule analyses. We suggest that the major function of the Z-ring is to govern nascent cell pole morphogenesis by directing the spatiotemporal distribution of septal cell wall remodeling enzymes through the Z-ring's GTP hydrolysis–dependent treadmilling dynamics. In this role, FtsZ functions in cell division as the counterpart of the cell shape–determining actin homolog MreB in cell elongation.
Expected final online publication date for the Annual Review of Biophysics, Volume 49 is May 6, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... This property has been explored in the design of compounds that fit the H-bonding pattern of 8-oxoG for its detection, [16] to control DNA structure, [17] in the formation of supramolecular helices with potential uses for electronic biodevices, [18] or in the use of C8-substituted guanosine analogues to understand biological mechanisms. [19] Of note is an example in which a DNA aptamer was designed to detect 8-oxoG through interactions similar to those shown in Scheme 1. [20] In addition, the presence of 8-oxoG has also been shown to affect the overall structure of DNA and to lead to its deformation. [21] These examples provide evidence that, although the effects of this oxidative modification on RNA have not been explored in detail, there is great potential in using it as a handle to control the structure and function of RNA. ...
Aptamers are attractive constructs due to their high affinity/selectivity towards a target. Herein, 8‐Oxo‐7,8‐dihydroguanosine (8‐oxoG) was used, due in part to its unique H‐bonding capabilities (Watson‐Crick or Hoogsteen), to expand the ‘RNA alphabet’. Its impact was explored on the RNA aptamer of theophylline by modifying its binding pocket at positions G11, G25, or G26. Structural probing, via RNase A and T1, showed that modification at G11 leads to a drastic structural change while the G25‐/G26‐modified analogues exhibited similar cleavage patterns as the canonical construct. The recognition properties towards three xanthine derivatives were then explored via thermophoresis. Modifying the aptamer at position‐G11 led to binding inhibition, however modification at G25 changed the selectivity towards theobromine (Kd ~ 160 μM), while displaying a poor affinity for theophylline (Kd > 1.5 mM). Overall, 8‐oxoG can have an impact on the structure of aptamers in a position dependent manner, leading to altered target selectivity.
... FtsZ was isolated as described [52] and ZapA was isolated using a His-tagged version after which the His-tag was removed by proteolysis as described [8]. Polymerization of FtsZ was studied by light scattering in 1200 µL polymerization buffer (50 mM HEPES, pH 7.5, 50 mM KCl, 5 mM MgCl 2 ), 3.4 µM FtsZ, and 6.8 µM ZapA at 30 • C. ...
Cell division in bacteria is initiated by the polymerization of FtsZ at midcell in a ring-like structure called the Z-ring. ZapA and other proteins assist Z-ring formation and ZapA binds ZapB, which senses the presence of the nucleoids. The FtsZ–ZapA binding interface was analyzed by chemical cross-linking mass spectrometry (CXMS) under in vitro FtsZ-polymerizing conditions in the presence of GTP. Amino acids residue K42 from ZapA was cross-linked to amino acid residues K51 and K66 from FtsZ, close to the interphase between FtsZ molecules in protofilaments. Five different cross-links confirmed the tetrameric structure of ZapA. A number of FtsZ cross-links suggests that its C-terminal domain of 55 residues, thought to be largely disordered, has a limited freedom to move in space. Site-directed mutagenesis of ZapA reveals an interaction site in the globular head of the protein close to K42. Using the information on the cross-links and the mutants that lost the ability to interact with FtsZ, a model of the FtsZ protofilament–ZapA tetramer complex was obtained by information-driven docking with the HADDOCK2.2 webserver.
... A number of closely related FtsZ crystal structures were currently available for drug design, including FtsZ from archaea (Oliva et al. 2004(Oliva et al. , 2007, Gram-positive (Leung et al. 2004;Läppchen et al. 2008;Raymond et al. 2009;Tan et al. 2012;Matsui et al. 2012) and Gram-negative (Mosyak et al. 2000;Cordell et al. 2003;Leung et al. 2004;Oliva et al. 2007;) bacteria. All FtsZ possessed similar amino sequences, and they were nearly identical in structure except for different C-terminal extensions. ...
FtsZ, an essential cytokinesis protein, was a highly promising target for antibacterial agents. Following up the identification of trisubstituted benzimidazoles targeting Mycobacterium tuberculosis FtsZ (Mtb FtsZ) in previous studies and in order to explore the possible binding mode of these analogues, in silico methodologies such as 3D-QSAR, ProFunc analysis, molecular docking and molecular dynamic simulation were performed. They corroborated well with each other and gave credence to the proposed binding mechanism of trisubstituted benzimidazoles in the interdomain cleft region of Mtb FtsZ. The benzimidazole scaffold and cyclohexyl group of trisubstituted benzimidazoles were orthogonal to each other in low energy state and inclosed in a most hydrophobic environment formed by residues from the C-terminal β sheets and H7 helix. The carbamate groups at the 5-position extended outward form the cleft cavity to the hydrophilic surface. The substituents at the 6-position fitted into the top portion of the cleft by directly interacting with the T7 loop. It was believed that the hydrophobic interactions and the polar contacts were major contributors to the stabilization of the ligand binding in the interdomain cleft. The binding mechanism provided useful clues to design new trisubstituted benzimidazoles inhibitors of Mtb FisZ with promising activity. The work presented here may even be expanded tremendously to screen novel Mtb FtsZ inhibitors with different scaffolds by using the molecular dynamic refined Mtb FtsZ structure and docking based 3D QSAR CoMFA.
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... Läppchen et al. identified C8-substituted GTP analogs which selectively inhibited FtsZ polymerization while not affecting Tubulin assembly [120]; these ana- logs 33 (Figure 8) were shown to be reversible competitive inhibitors of GTP-driven polymerization of E. coli FtsZ (Table 6) [121]. However, C8-substituted analogs lack antibacterial activity (probably due to poor penetration across the bacterial cell envelope) [122]. ...
Filamenting temperature-sensitive mutant Z (FtsZ), an essential cell division protein in bacteria, has recently emerged as an important and exploitable antibacterial target. Cytokinesis in bacteria is regulated by the assembly dynamics of this protein, which is ubiquitously present in eubacteria. The perturbation of FtsZ assembly has been found to have a deleterious effect on the cytokinetic machinery and, in turn, upon cell survival. FtsZ is highly conserved among prokaryotes, offering the possibility of broad-spectrum antibacterial agents, while its limited sequence homology with tubulin (an essential protein in eukaryotic mitosis) offers the possibility of selective toxicity. This review aims to summarize current knowledge regarding the mechanism of action of FtsZ, and to highlight existing attempts toward the development of clinically useful inhibitors.
... FtsZ has been validated as the target of the in vivo effective antibacterial compound PC190723 (7), which modulates FtsZ assembly by stabilizing its polymers (8,9) and binds into the cleft between the C-terminal domain and the core helix H7 of this protein (9)(10)(11). Moreover, several nucleotide analogs (12,13) and nonnucleotide compounds that selectively target the nucleotide site of FtsZ have also been identified (14)(15)(16). ...
The cytoskeletal protein FtsZ assembles in a head-to-tail manner, forming dynamic filaments that are essential for bacterial cell division. Our studies using unbiased molecular dynamics simulations from representative filament crystal structures disclose different filament curvatures supported by nucleotide-regulated hinge-bending motions between consecutive FtsZ subunits, in agreement with experimental observations, and unravel the natural mechanism of the FtsZ assembly switch. Whereas GTP-FtsZ filaments bend and twist in a preferred orientation, thereby occluding the nucleotide binding site, the differently curved GDP-FtsZ filaments identified exhibit a heterogeneous distribution of open and closed interfaces between monomers.
... On the other hand, C8-substituted GTP analogs inhibit FtsZ polymerization while supporting tubulin assembly, confirming that selective inhibition of the GTP-binding site is possible without unwanted side effects in eukaryotic tubulin. 27 However, these GTP derivatives lack antibacterial activity probably due to poor penetration across the bacterial cell envelope. ...
Essential cell division protein FtsZ is considered an attractive target in the search for antibacterials with novel mechanisms of action to overcome the resistance problem. FtsZ undergoes GTP-dependent assembly at mid-cell to form the Z-ring, a dynamic structure that evolves until final constriction of the cell. Therefore, molecules able to inhibit its activity will eventually disrupt bacterial viability. In this work, we report a new series of small molecules able to replace GTP and to specifically inhibit FtsZ, blocking the bacterial division process. These new synthesized inhibitors interact with the GTP-binding site of FtsZ (Kd = 0.4-0.8 μM), display antibacterial activity against Gram-positive pathogenic bacteria, and show selectivity against tubulin. Biphenyl derivative 28 stands out as a potent FtsZ inhibitor (Kd = 0.5 µM) with high antibacterial activity [MIC (MRSA) = 7 μM]. In-depth analysis of the mechanism of action of compounds 22, 28, 33 and 36, has revealed that they act as effective inhibitors of correct FtsZ assembly, blocking bacterial division and thus leading to filamentous undivided cells. These findings provide a compelling rationale for the development of compounds targeting the GTP-binding site as antibacterial agents and open the door to antibiotics with novel mechanisms of action.
... FtsZ has been validated as the target of the in vivo effective antibacterial compound PC190723 (7), which modulates FtsZ assembly by stabilizing its polymers (8,9) and binds into the cleft between the C-terminal domain and the core helix H7 of this protein (9)(10)(11). Moreover, several nucleotide analogs (12,13) and nonnucleotide compounds that selectively target the nucleotide site of FtsZ have also been identified (14)(15)(16). ...
Abstract Bacterial cytoskeletal protein FtsZ assembles in a head-to-tail manner, forming dynamic filaments that are essential for cell division. Here, we study their dynamics using unbiased atomistic molecular simulations from representative filament crystal structures. In agreement with experimental data, we find different filament curvatures that are supported by a nucleotide-regulated hinge motion between consecutive FtsZ monomers. Whereas GTP-FtsZ filaments bend and twist in a preferred orientation, thereby burying the nucleotide, the differently curved GDP-FtsZ filaments exhibit a heterogeneous distribution of open and closed interfaces between monomers. We identify a coordinated Mg2+ ion as the key structural element in closing the nucleotide site and stabilizing GTP filaments, whereas the loss of the contacts with loop T7 from the next monomer in GDP filaments leads to open interfaces that are more prone to depolymerization. We monitored the FtsZ monomer assembly switch, which involves opening/closing of the cleft between the C-terminal domain and the H7 helix, and observed the relaxation of isolated and filament minus-end monomers into the closed-cleft inactive conformation. This result validates the proposed switch between the low-affinity monomeric closed-cleft conformation and the active open-cleft FtsZ conformation within filaments. Finally, we observed how the antibiotic PC190723 suppresses the disassembly switch and allosterically induces closure of the intermonomer interfaces, thus stabilizing the filament. Our studies provide detailed structural and dynamic insights into modulation of both the intrinsic curvature of the FtsZ filaments and the molecular switch coupled to the high-affinity end-wise association of FtsZ monomers.
... A number of small molecule inhibitors of FtsZ have already been shown to prevent FtsZ polymerization and inhibit bacterial cell division [14][15][16][17][18][19][20]. The molecules bind to one of two alternative sites of FtsZ ( Figure 1A): at the N-terminal GTP binding site [21][22][23], or at the C-terminal interdomain cleft [24]. Compounds targeting the highly conserved GTP binding site mimic the natural substrate of the enzyme and might have potential advantages for developing broad-spectrum antibacterial agents [25]. ...
Inhibition of the functional activity of Filamenting temperature-sensitive mutant Z (FtsZ) protein, an essential and highly conserved bacterial cytokinesis protein, is a promising approach for the development of a new class of antibacterial agents. Berberine, a benzylisoquinoline alkaloid widely used in traditional Chinese and native American medicines for its antimicrobial properties, has been recently reported to inhibit FtsZ. Using a combination of in silico structure-based design and in vitro biological assays, 9-phenoxyalkyl berberine derivatives were identified as potent FtsZ inhibitors. Compared to the parent compound berberine, the derivatives showed a significant enhancement of antibacterial activity against clinically relevant bacteria, and an improved potency against the GTPase activity and polymerization of FtsZ. The most potent compound 2 strongly inhibited the proliferation of Gram-positive bacteria, including methicillin-resistant S. aureus and vancomycin-resistant E. faecium, with MIC values between 2 and 4 µg/mL, and was active against the Gram-negative E. coli and K. pneumoniae, with MIC values of 32 and 64 µg/mL respectively. The compound perturbed the formation of cytokinetic Z-ring in E. coli. Also, the compound interfered with in vitro polymerization of S. aureus FtsZ. Taken together, the chemical modification of berberine with 9-phenoxyalkyl substituent groups greatly improved the antibacterial activity via targeting FtsZ.
... Crystal structure of FtsZ from M. jannaschii with GDP bound (PDB entry 1FSZ, Löwe and Amos, 1998). Thus far no differences have been observed between the GTP and the GDP bound structures of FtsZ (Läppchen et al., 2008) immediately hydrolysed . The active site for GTP is formed at the interface of two FtsZ subunits where one subunit provides the binding pocket for GTP and the other the cation-co-ordinating T7 loop (Löwe and Amos, 1999;. ...
... S15A) (8). The same T-state conformation is maintained in all GTPbound and some GDP-bound FtsZ structures (7,19,20) and is necessary for the longitudinal assembly of a straight FtsZ protofilament (5), in which the T3 loop interacts extensively with the T7 loop of the top subunit. In contrast, in our GDP-bound MtbFtsZ structure, the T3 loop adopts a relaxed conformation (R state) in the absence of the g-phosphate ( fig. ...
The essential bacterial protein FtsZ is a guanosine triphosphatase that self-assembles into a structure at the division site
termed the “Z ring”. During cytokinesis, the Z ring exerts a constrictive force on the membrane by using the chemical energy
of guanosine triphosphate hydrolysis. However, the structural basis of this constriction remains unresolved. Here, we present
the crystal structure of a guanosine diphosphate–bound Mycobacterium tuberculosis FtsZ protofilament, which exhibits a curved conformational state. The structure reveals a longitudinal interface that is
important for function. The protofilament curvature highlights a hydrolysis-dependent conformational switch at the T3 loop
that leads to longitudinal bending between subunits, which could generate sufficient force to drive cytokinesis.
... Amongst the model organisms for bacterial cell division, the CTL of B. subtilis and E. coli is ~50 residues, while that of Caulobacter crescentus, a member of the Alpha-Proteobacteria, is 176. Notably, the CTL is irresolvable on crystal structures of bacterial FtsZ (Leung et al., 2004;Oliva et al., 2007;Haydon et al., 2008;Läppchen et al., 2008;Raymond et al., 2009 ;Matsui et al., 2012), and therefore has been presumed to be an intrinsically disordered peptide (IDP) (Erickson et al., 2010). Previous work suggests the CTL is flexible with a contour length of 17 nm, and an average end-to-end distance of 5.2 nm for the relaxed peptide (Ohashi et al., 2007). ...
Assembly of the cytoskeletal protein FtsZ into a ring-like structure is required for bacterial cell division. Structurally, FtsZ consists of four domains: the globular N-terminal core, a flexible linker, 8-9 conserved residues implicated in interactions with modulatory proteins, and a highly variable set of 4-10 residues at its very C terminus. Largely ignored and distinguished by lack of primary sequence conservation, the linker is presumed to be intrinsically disordered. Here we employ genetics, biochemistry and cytology to dissect the role of the linker in FtsZ function. Data from chimeric FtsZs substituting the native linker with sequences from unrelated FtsZs as well as a helical sequence from human beta-catenin indicate that while variations in the primary sequence are well tolerated, an intrinsically disordered linker is essential for B. subtilis FtsZ assembly. Linker lengths ranging from 25-100 residues supported FtsZ assembly, but replacing the B. subtilis FtsZ linker with a 249-residue linker from A. tumefaciens FtsZ interfered with cell division. Overall, our results support a model in which the linker acts as a flexible tether allowing FtsZ to associate with the membrane through a conserved C-terminal domain while simultaneously interacting with itself and modulatory proteins in the cytoplasm.
In the pre-antibiotic era, common bacterial infections accounted for high mortality and morbidity. Moreover, the discovery of penicillin in 1928 marked the beginning of an antibiotic revolution, and this antibiotic era witnessed the discovery of many novel antibiotics, a golden era. However, the misuse or overuse of these antibiotics, natural resistance that existed even before the antibiotics were discovered, genetic variations in bacteria, natural selection, and acquisition of resistance from one species to another consistently increased the resistance to the existing antibacterial targets. Antibacterial resistance (ABR) is now becoming an ever-increasing concern jeopardizing global health. Henceforth, there is an urgent unmet need to discover novel compounds to combat ABR, which act through untapped pathways/ mechanisms. Filamentous Temperature Sensitive mutant Z (FtsZ) is one such unique target, a tubulin homolog involved in developing a cytoskeletal framework for the cytokinetic ring. Additionally, its pivotal role in bacterial cell division and the lack of homologous structural protein in mammals makes it a potential antibacterial target for developing novel molecules. Approximately 2176 X-crystal structures of FtsZ were available, which initiated the research efforts to develop novel antibacterial agents. The literature has reported several natural, semisynthetic, peptides, and synthetic molecules as FtsZ inhibitors. This review provides valuable insights into the basic crystal structure of FtsZ, its inhibitors, and their inhibitory activities. This review also describes the available in vitro detection and quantification methods of FtsZ-drug complexes and the various approaches for determining drugs targeting FtsZ polymerization.
Small molecules as antibacterial agents have contributed immensely to the growth of modern medicine over the last several decades. However, the emergence of drug resistance among bacterial pathogens has undermined the effectiveness of the existing antibiotics. Thus, there is an exigency to address the antibiotic crisis by developing new antibacterial agents and identifying novel drug targets in bacteria. In this review, we summarize the importance of guanosine triphosphate hydrolyzing proteins (GTPases) as key agents for bacterial survival. We also discuss representative examples of small molecules that target bacterial GTPases as novel antibacterial agents, and highlight areas that are ripe for exploration. Given their vital roles in cell viability, virulence, and antibiotic resistance, bacterial GTPases are highly attractive antibacterial targets that will likely play a vital role in the fight against antimicrobial resistance.
The rapid emergence of antibiotic resistance has become a prevalent threat to public health, thereby development of new antibacterial agents having novel mechanisms of action is in an urgent need. Targeting at the cytoskeletal cell division protein filamenting temperature-sensitive mutant Z (FtsZ) has been validated as an effective and promising approach for antibacterial drug discovery. In this study, a series of novel biphenyl-benzamides as FtsZ inhibitors has been rationally designed, synthesized and evaluated for their antibacterial activities against various Gram-positive bacteria strains. In particular, the most promising compound 30 exhibited excellent antibacterial activities, especially against four different Bacillus subtilis strains, with an MIC range of 0.008 μg/mL to 0.063 μg/mL. Moreover, compound 30 also showed good pharmaceutical properties with low cytotoxicity (CC50 > 20 μg/mL), excellent human metabolic stability (T1/2 = 111.98 min), moderate pharmacokinetics (T1/2 = 2.26 h, F = 61.2%) and in vivo efficacy, which can be identified as a promising FtsZ inhibitor worthy of further profiling.
Currently, wide-spread antimicrobials resistance among bacterial pathogens continues being a dramatically increasing and serious threat to public health, and thus there is a pressing need to develop new antimicrobials to keep pace with the bacterial resistance. Filamentous temperature-sensitive protein Z (FtsZ), a prokaryotic cytoskeleton protein, plays an important role in bacterial cell division. It as a very new and promising target, garners special attention in the antibacterial research in the recent years. This review describes not only the function and dynamic behaviors of FtsZ, but also the known natural and synthetic inhibitors of FtsZ. In particular, the small molecules recently developed and the future directions of ideal candidates are highlighted.
The rise of antibiotic-resistant infections has been well documented and the need for novel antibiotics cannot be overemphasized. US FDA approved antibiotics target only a small fraction of bacterial cell wall or membrane components, well-validated antimicrobial targets. In this review, we highlight small molecules that inhibit relatively unexplored cell wall and membrane targets. Some of these targets include teichoic acids-related proteins (DltA, LtaS, TarG and TarO), lipid II, Mur family enzymes, components of LPS assembly (MsbA, LptA, LptB and LptD), penicillin-binding protein 2a in methicillin-resistant Staphylococcus aureus, outer membrane protein transport (such as LepB and BamA) and lipoprotein transport components (LspA, LolC, LolD and LolE). Inhibitors of SecA, cell division protein, FtsZ and compounds that kill persister cells via membrane targeting are also covered.
Cystic fibrosis is a rare genetic disease characterized by the production of dehydrated mucus in the lung able to trap bacteria and rendering their proliferation particularly dangerous, thus leading to chronic infections. Among these bacteria, Staphylococcus aureus and Pseudomonas aeruginosa play a major role while, within emerging pathogens, Stenotrophomonas maltophilia, Achromobacter xylosoxidans, Burkholderia cepacia complex species, as well as non-tuberculous mycobacteria are listed. Since a common feature of these bacteria is the high level of drug resistance, cell division, and in particular FtsZ, has been explored as a novel therapeutic target for the design of new molecules with antibacterial properties. This review summarizes and provides insight into recent advances in the discovery of compounds targeting FtsZ: the majority of them exhibit anti-staphylococcal activity, while a few were directed against the cystic fibrosis Gram negative pathogens.
The essential bacterial division protein FtsZ uses GTP binding and hydrolysis to assemble into dynamic filaments that treadmill around the Z‐ring, guiding septal wall synthesis and cell division. FtsZ is a structural homolog of tubulin and a target for discovering new antibiotics. Here, using FtsZ from the pathogen S aureus (SaFtsZ), we reveal that, prior to assembly, FtsZ monomers require nucleotide binding for folding; this is possibly relevant to other mesophilic FtsZs. Apo‐SaFtsZ is essentially unfolded, as assessed by nuclear magnetic resonance and circular dichroism. Binding of GTP (≥ 1mM) dramatically shifts the equilibrium towards the active folded protein. Supportingly, SaFtsZ refolded with GDP crystallizes in a native structure. Apo‐SaFtsZ also folds with 3.4 M glycerol, enabling high‐affinity GTP binding (KD 20 nM determined by isothermal titration calorimetry) similar to thermophilic stable FtsZ. Other stabilizing agents that enhance nucleotide binding include ethylene glycol, trimethylamine N‐oxide (TMAO), and several bacterial osmolytes. High salt stabilizes SaFtsZ without bound nucleotide in an inactive twisted conformation. We identified a cavity behind the SaFtsZ‐GDP nucleotide‐binding pocket that harbors different small compounds, which is available for extended nucleotide‐replacing inhibitors. Furthermore, we devised a competition assay to detect any inhibitors that overlap the nucleotide site of SaFtsZ, or Escherichia coli FtsZ, employing osmolyte‐stabilized apo‐FtsZs and the specific fluorescence anisotropy change of mant‐GTP upon dissociation from the protein. This robust assay provides a basis to screening for high affinity GTP‐replacing ligands, which combined with structural studies and phenotypic profiling should facilitate development of a next generation of FtsZ‐targeting antibacterial inhibitors.
Alchemical free energy calculations using conventional molecular dynamics and thermodynamic integration rely on simulations performed at fixed values of the coupling parameter λ. When multiple conformers at equilibrium are separated by high barriers in the space orthogonal to λ, proper convergence may require extremely long simulations. Four main strategies can be employed to address this orthogonal-sampling problem: (a) λ-variations, where λ can change along the simulations to circumvent barriers; (b) λ-extrapolations, where statistical information is transferred between λ-points; (c) specific biasing, where orthogonal barriers are reduced using a biasing potential designed specifically for the system; (d) generic biasing, where orthogonal barriers are reduced using a generic approach. Here, we investigate the relative merits of the first three strategies considering two benchmark systems: the KXK system involves a mutation of the central residue in a tripeptide to a glycine, and the XTP system involves a hydrogen-to-bromine mutation in the base of a nucleotide. Three sampling methods are compared, the latter two involving λ-variations: conventional MD simulations, Hamiltonian replica exchange, and the recently introduced conveyor belt method. Two free energy estimators are applied, the second one involving λ-extrapolations: TI with Simpson quadrature and the multistate Bennett acceptance ratio. Finally, three different seeding schemes are considered for the generation of the initial configurations. For both benchmark systems, λ-extrapolations are found to provide little gain, whereas λ-variations significantly enhance the convergence. λ-variations are sufficient on their own if the orthogonal barriers are low in at least one state (e.g., the glycine state in KXK). However, if the orthogonal barriers are high over the entire λ-range (e.g., the XTP system), λ-variations are only effective when adding a specific biasing.
The continuous emergence and rapid spread of a multidrug-resistant strain of bacterial pathogens have demanded the discovery and development of new antibacterial agents. A highly conserved prokaryotic cell division protein FtsZ is considered as a promising target by inhibiting bacterial cytokinesis. Inhibition of FtsZ assembly restrains the cell-division complex known as divisome, which results in filamentation, leading to lysis of the cell. This review focuses on details relating to the structure, function, and influence of FtsZ in bacterial cytokinesis. It also summarizes on the recent perspective of the known natural and synthetic inhibitors directly acting on FtsZ protein, with prominent antibacterial activities. A series of benzamides, trisubstituted benzimidazoles, isoquinolene, guanine nucleotides, zantrins, carbonylpyridine, 4 and 5-Substituted 1-phenyl naphthalenes, sulindac, vanillin analogues were studied here and recognized as FtsZ inhibitors that act either by disturbing FtsZ polymerization and/or GTPase activity. Doxorubicin, from a U.S. FDA, approved drug library displayed strong interaction with FtsZ. Several of the molecules discussed, include the prodrugs of benzamide based compound PC190723 (TXA-709 and TXA707). These molecules have exhibited the most prominent antibacterial activity against several strains of Staphylococcus aureus with minimal toxicity and good pharmacokinetics properties. The evidence of research reports and patent documentations on FtsZ protein has disclosed distinct support in the field of antibacterial drug discovery. The pressing need and interest shall facilitate the discovery of novel clinical molecules targeting FtsZ in the upcoming days.
The disturbing increase in the number of bacterial pathogens that are resistant to multiple, or sometimes all, current antibiotics highlights the desperate need to pursue the discovery and development of novel classes of antibacterials. The wealth of knowledge available about the bacterial cell division machinery has aided target-driven approaches to identify new inhibitor compounds. The main division target being pursued is the highly conserved and essential protein, FtsZ. Despite very active research on FtsZ inhibitors for several years, this protein is not yet targeted by any commercial antibiotic. Here we discuss the suitability of FtsZ as an antibacterial target for drug development, and review progress achieved in this area. We use hindsight to highlight the gaps that have slowed progress in FtsZ inhibitor development, and to suggest guidelines for concluding that FtsZ is actually the target of these molecules, a key missing link in several studies. In moving forward, a multidisciplinary and a communicative and collaborative process, with sharing of research expertise, is critical if we are to succeed.
The bacterial cell division protein FtsZ is conserved in most bacteria and essential for viability. There have been concerted efforts in developing inhibitors that target FtsZ as potential antibiotics. Key to this is an in-depth understanding of FtsZ structure at the molecular level across diverse bacterial species to ensure inhibitors have high affinity for the FtsZ target in a variety of clinically relevant pathogens. In this study, we show that FtsZ structures differ in three ways: (1) the H7 helix curvature; (2) the dimensions of the interdomain cleft; and (3) the opening/closing mechanism of the interdomain cleft, whereas no differences were observed in the dimensions of the nucleotide-binding pocket and T7 loop. Molecular dynamics simulation may suggest that there are two possible mechanisms for the process of opening and closing of the interdomain cleft on FtsZ structures. This discovery highlights significant differences between FtsZ structures at the molecular level and this knowledge is vital in assisting the design of potent FtsZ inhibitors.
FtsZ, a homolog of tubulin, is found in almost all bacteria and archaea where it has a primary role in cytokinesis. Evidence for structural homology between FtsZ and tubulin came from their crystal structures and identification of the GTP-box. Tubulin and FtsZ constitute a distinct family of GTPases and show striking similarities in many of their polymerization properties. The differences between them, more so, the complexities of microtubule dynamic behavior in comparison to that of FtsZ, indicate that the evolution to tubulin is attributable to the incorporation of the complex functionalities in higher organisms. FtsZ and microtubules function as polymers in cell division but their roles differ in the division process. The structural and partial functional homology has made the study of their dynamic properties more interesting. In this review, we focus on the application of the information derived from studies on FtsZ dynamics to study microtubule dynamics and vice versa. The structural and functional aspects that led to the establishment of the homology between the two proteins are explained to emphasize the network of FtsZ and microtubule studies and how they are connected.
FtsZ is an essential protein for bacterial cell division, and an attractive and underexploited novel antibacterial target protein. Screening of Indonesian plants revealed the inhibitory activity of the methanol extract of Glycyrrhiza glabra on the Bacillus subtilis FtsZ (BsFtsZ) GTPase, and further bioassay-guided fractionation of the active methanol extract led to the isolation of seven known polyketides (1-7). Among them, gancaonin I (1), glycyrin (3), and isolicoflavanol (5) exhibited anti-BsFtsZ GTPase activities, at levels comparable to that of the synthetic FtsZ inhibitor, Zantrin Z3. Enzymatic assays using a BsFtsZ Val307R mutant protein and in silico simulations suggested that 1, 3, and 5 bind to the cleft on BsFtsZ, as in the case of the previously reported uncompetitive FtsZ inhibitor, PC190723, and thereby display their significant anti-BsFtsZ inhibitory activities. Furthermore, 1 also showed significant inhibitory activity against B. subtilis, with a MIC value of 5μM. The present study provides new insights into the naturally occurring B. subtilis growth inhibitors.
FtsZ is a widely conserved tubulin-like GTPase that directs bacterial cell division and a new target for antibiotic discovery. This protein assembly machine cooperatively polymerizes forming single-stranded filaments, by means of self-switching between inactive and actively associating monomer conformations. The structural switch mechanism was proposed to involve a movement of the C-terminal and N-terminal FtsZ domains, opening a cleft between them, allosterically coupled to the formation of a tight association interface between consecutive subunits along the filament. The effective antibacterial benzamide PC190723 binds into the open interdomain cleft and stabilizes FtsZ filaments, thus impairing correct formation of the FtsZ ring for cell division. We have designed fluorescent analogs of PC190723 to probe the FtsZ structural assembly switch. Among them, nitrobenzoxadiazole probes specifically bind to assembled FtsZ rather than to monomers. Probes with several spacer lengths between the fluorophore and benzamide moieties suggest a binding site extension along the interdomain cleft. These probes label FtsZ rings of live Bacillus subtilis and Staphylococcus aureus, without apparently modifying normal cell morphology and growth, but at high concentrations they induce impaired bacterial division phenotypes typical of benzamide antibacterials. During the FtsZ assembly-disassembly process, the fluorescence anisotropy of the probes changes upon binding and dissociating from FtsZ, thus reporting open and closed FtsZ interdomain clefts. Our results demonstrate the structural mechanism of the FtsZ assembly switch, and suggest that the probes bind into the open clefts in cellular FtsZ polymers preferably to unassembled FtsZ in the bacterial cytosol.
Novel antibiotics are urgently needed to combat the rise of infections due to drug-resistant microorganisms. Numerous natural nucleosides and their synthetically modified analogues have been reported to have moderate to good antibiotic activity against different bacterial and fungal strains. Nucleoside-based compounds target several crucial processes of bacterial and fungal cells such as nucleoside metabolism and cell wall, nucleic acid and protein biosynthesis. Nucleoside analogues have also been shown to target many other bacterial and fungal cellular processes though these are not well characterised and may therefore represent opportunities to discover new drugs with unique mechanisms of action. In this perspective we demonstrate that nucleoside analogues, cornerstones of anticancer and antiviral treatments, also have great potential to be repurposed as antibiotics so that an old drug can learn new tricks.
A straight forward strategy to synthesize purine nucleosides with multiple functionalization on 2-, 6-, and 8-positions has been developed successfully, which provides a series of 8-alkoxy-6-alkylamino-2-alkylthiopurine nucleosides in moderate to good yields for further biological and medical activities screening.
Similar to its eukaryotic counterpart, the prokaryotic cytoskeleton is essential for the structural and mechanical properties of bacterial cells. The essential protein FtsZ is a central player in the cytoskeletal family, forms a cytokinetic ring at mid-cell, and recruits the division machinery to orchestrate cell division. Cells depleted of or lacking functional FtsZ do not divide and grow into long filaments that eventually lyse. FtsZ has been studied extensively as a target for antibacterial development. In this perspective, we review the structural and biochemical properties of FtsZ, its role in cell biochemistry and physiology, the different mechanisms of inhibiting FtsZ, small molecule antagonists (including some misconceptions about mechanisms of action), and their discovery strategies. This collective information will inform chemists on different aspects of FtsZ that can be - and have been - used to develop successful strategies for devising new families of cell division inhibitors.
Eukaryotic αβ-tubulin and bacterial FtsZ self-assemble into dynamic cytoskeletal polymers, microtubules or filaments, which are essential for chromosome segregation or bacterial cell division, respectively. Both share homologous core structures with guanosine-5'-triphosphate (GTP)-binding and GTPase-activating domains joined by a central helix, and form similar protofilaments with 4 nm spaced subunits along them. During assembly, the GTPase-activating domain of one subunit associates with the GTP binding domain of the preceding subunit, completing the active site. GTP hydrolysis triggers disassembly, which is coupled to free subunits switching into inactive conformation. Microtubule dynamics is inhibited by anticancer drugs binding near tubulin assembly interfaces. FtsZ is a target for new antibiotics discovery; several bacterial division inhibitors bind between FtsZ domains or at its GTP site. Other proteins in this superfamily include: gamma-tubulin that is essential for microtubule organisation; bacterial tubulin, a primitive structural homologue; and recently discovered TubZ, distant homologue employed by plasmids and phages for deoxyribonucleic acid positioning.
Nucleoside analogues have attracted much attention due to their potential biological activities. Amongst all synthetic nucleosides, C5-modified pyrimidines and C2-or C8-modified purines have been particularly studied. A large variety of palladium cross-coupling reactions, with a majority of them based on the Suzuki-Miyaura reaction, have been developed for preparing the desired nucleoside derivatives. Our objective is to focus this review on the Suzuki-Miyaura cross-coupling of nucleosides using methodologies compatible with green chemistry and sustainable development for one part and bioorthogonality for the other part, which means using aqueous medium and no protection/deprotection steps.
We demonstrate that multivalent, polymeric 8-methoxyguanosine derivatives based on poly(dimethylacrylamide) can enhance the mechanical properties of the low molecular weight hydrogelator 8-methoxy-2′,3′,5′-tri-O-acetylguanosine at biologically relevant salt concentrations. It is proposed that these nongelling polymeric derivatives, under the conditions studied, can result in a significant enhancement of these supramolecular gels (e.g., for gels containing 1 wt % gelator G′ can be increased from ca. 2000 Pa with no additive to 80 000 Pa) by acting as supramolecular cross-linking units. Two competing mechanisms appear to play a role in these cogels. At low polymer concentrations the guanosine-containing polymers tend to act more as solubilizing agents for the gelator, thus weakening the gels, while at high guanosine-containing polymer concentrations the gels show a marked enhancement in mechanical properties consistent with them acting as supramolecular cross-linking agents. As such, the thermomechanical properties of these cogels depend on both the polymer:low molecular weight gelator ratio and the number of 8-methoxyguanosine repeat units present in the polymer additive. Thus, these polymeric guanosine-based additives impart the ability to tailor both the modulus and shear sensitivity of the gels. For example, cogels with a modulus ranging between ca. 95 and 80 000 Pa can be obtained through judicious selection of the type and amount of polymer additive.
The conformational preference of the O6-benzyl-guanine (BzG) adduct was computationally examined using nucleoside, nucleotide and DNA models, which provided critical information about the potential mutagenic consequences and toxicity of the BzG adduct in our cells. Substantial conformational flexibility of the BzG moiety, including rotation of the bulky group with respect to the base and the internal conformation of the bulk moiety, is seen in the nucleoside and nucleotide models. This large conformational flexibility suggests the conformation adopted by BzG is dependent on the local environment of the BzG adduct. Upon incorporation of the adduct into the DNA helix, the BzG conformational flexibility is maintained. The range of BzG conformations adopted in DNA likely arises due to a combination of the long and flexible (-CH2¬-) linker, the small adduct size and the lack of discrete interactions between the bulky moiety and G. Due to the conformational flexibility of the adduct, many DNA conformations are observed for BzG adducted DNA, including those not previously reported in the literature, and thus a modified nomenclature for adducted DNA conformations is presented. Furthermore, the preferred conformation of BzG adducted DNA is greatly dependent on a number of factors, including the pairing nucleotide, the discrete interactions in the helix and the solvation of the benzyl moiety. These factors in turn lead to a complicated mutagenic and toxic profile that may invoke pairing with natural C, mispairs, or deletion mutations, which is supported by previously reported experimental biochemical studies. Despite this complex mutagenic profile, pairing with C leads to the most stable helical structure, which is the first combined structural and energetic explanation for experimental studies reporting a higher rate of C incorporation than any other nucleobase upon the BzG replication.
Cell division protein FtsZ is the organizer of the cytokinetic Z-ring in most bacteria and a target for new antibiotics. FtsZ assembles with GTP into filaments that hydrolyze the nucleotide at the association interface between monomers and then disassemble. We have replaced FtsZ´s GTP with non-nucleotide synthetic inhibitors of bacterial division. We searched for these small molecules among compounds from the literature, from virtual screening (VS) and from our in-house synthetic library (UCM), employing a fluorescence anisotropy primary assay. From these screens we have identified the polyhydroxy aromatic compound UCM05 and its simplified analog UCM44 that specifically bind to Bacillus subtilis FtsZ monomers with micromolar affinities and perturb normal assembly, as examined with light scattering, polymer sedimentation and negative stain electron microscopy. On the other hand, these ligands induce the cooperative assembly of nucleotide-devoid archaeal FtsZ into distinct well-ordered polymers, different from GTP-induced filaments. These FtsZ inhibitors impair localization of FtsZ into the Z-ring and inhibit bacterial cell division. The chlorinated analog UCM53 inhibits the growth of clinical isolates of antibiotic-resistant Staphylococcus aureus and Enterococcus faecalis. We suggest that these interfacial inhibitors recapitulate binding and some assembly-inducing effects of GTP, but impair the correct structural dynamics of FtsZ filaments and thus inhibit bacterial division, possibly by binding to a small fraction of the FtsZ molecules in a bacterial cell, which opens a new approach to FtsZ-based antibacterial drug discovery.
Direct alkylation via a free radical reaction was utilized to prepare 8-tert-butylguanosine-5′- phosphate; this was dephosphorylated with alkaline phosphatase to give the parent nucleoside. The 8- (α-hydroxyisopropyl) derivatives of 5′-GMP and guanosine were prepared in an analogous manner.
The foregoing products, which are necessarily in the syn conformation about the glycosidic bond because of the bulky 8-substituents (van der Waals’ radii 3.5-4 Å), were characterized by elementary analysis, UV spectra and ¹ HNMR spectroscopy. The changes in chemical shifts of H (1′), H (2′) and H (3′) of each of the foregoing derivatives, relative to those for guanosine and 5′-GMP, were consistent with their being in the syn conformation and, furthermore, pointed to guanosine and 5′-GMP being predominantly anti, in aqueous medium or DMSO. They also demonstrated that, contrary to the prevailing opinion, 8-bromo-5′-GMP and 8-bromoguanosine are not necessarily exclusively in the syn conformation in solution.
While 8-bromoadenosine-5′-phosphate was slowly dephosphorylated by snake venom 5′-nucleoti- dase, the 8-tert-butyl and 8-(α-hydroxyisopropyl) derivatives of 5′-GMP were fully resistant to this enzyme.
We present a refined model of the αβ-tubulin dimer to 3.5 Å resolution. An improved experimental density for the zinc-induced tubulin sheets was obtained by adding 114 electron diffraction patterns at 40-60 ° tilt and increasing the completeness of structure factor amplitudes to 84.7 %. The refined structure was obtained using maximum-likelihood including phase information from experimental images, and simulated annealing Cartesian refinement to an R-factor of 23.2 and free R-factor of 29.7. The current model includes residues α:2-34, α:61-439, β:2-437, one molecule of GTP, one of GDP, and one of taxol, as well as one magnesium ion at the non-exchangeable nucleotide site, and one putative zinc ion near the M-loop in the α-tubulin subunit. The acidic C-terminal tails could not be traced accurately, neither could the N-terminal loop including residues 35-60 in the α-subunit. There are no major changes in the overall fold of tubulin with respect to the previous structure, testifying to the quality of the initial experimental phases. The overall geometry of the model is, however, greatly improved, and the position of side-chains, especially those of exposed polar/charged groups, is much better defined. Three short protein sequence frame shifts were detected with respect to the non-refined structure. In light of the new model we discuss details of the tubulin structure such as nucleotide and taxol binding sites, lateral contacts in zinc-sheets, and the significance of the location of highly conserved residues.
Tubulin and FtsZ share a common fold of two domains connected by a central helix. Structure-based sequence alignment shows that common residues localize in the nucleotide-binding site and a region that interacts with the nucleotide of the next tubulin subunit in the protofilament, suggesting that tubulin and FtsZ use similar contacts to form filaments. Surfaces that would make lateral interactions between protofilaments or interact with motor proteins are, however, different. The highly conserved nucleotide-binding sites of tubulin and FtsZ clearly differ from those of EF-Tu and other GTPases, while resembling the nucleotide site of glyceraldehyde-3-phosphate dehydrogenase. Thus, tubulin and FtsZ form a distinct family of GTP-hydrolyzing proteins.
The capacity of the centrosome to influence the lattice structure of nucleated microtubules was studied in vitro. Brain microtubules self-assembled to give predominantly (98%) 14-protofilament microtubules. However, under exactly the same conditions of assembly they grew off of purified centrosomes from neuroblastoma cells to give mostly (82%) 13-protofilament microtubules. Thus, the nucleation sites on the centrosome constrained the microtubule lattice to yield the number of protofilaments usually found in vivo.
FtsZ is an essential cell division protein in Escherichia coli that forms a ring structure at the division site under cell cycle control. The dynamic nature of the FtsZ ring suggests possible similarities to eukaryotic filament forming proteins such as tubulin. In this study we have determined that FtsZ is a GTP/GDP binding protein with GTPase activity. A short segment of FtsZ is homologous to a segment in tubulin believed to be involved in the interaction between tubulin and guanine nucleotides. A lethal ftsZ mutation, ftsZ3 (Rsa), that leads to an amino acid alteration in this homologous segment decreased GTP binding and hydrolysis, suggesting that interaction with GTP is essential for ftsZ function.
Bacterial cell division ends with septation, the constriction of the cell wall and cell membranes that leads to the formation of two daughter cells. During septation, FtsZ, a protein of relative molecular mass 40,000 which is ubiquitous in eubacteria and is also found in archaea and chloroplasts, localizes early at the division site to form a ring-shaped septum. This septum is required for the mechanochemical process of membrane constriction. FtsZ is a GTPase with weak sequence homology to tubulins. The nature of FtsZ polymers in vivo is unknown, but FtsZ can form tubules, sheets and minirings in vitro. Here we report the crystal structure at 2.8 A resolution of recombinant FtsZ from the hyperthermophilic methanogen Methanococcus jannaschii. FtsZ has two domains, one of which is a GTPase domain with a fold related to one found in the proteins p21ras and elongation factor EF-Tu. The carboxy-terminal domain, whose function is unknown, is a four-stranded beta-sheet tilted by 90 degrees against the beta-sheet of the GTPase domain. The two domains are arranged around a central helix. GDP binding is different from that typically found in GTPases and involves four phosphate-binding loops and a sugar-binding loop in the first domain, with guanine being recognized by residues in the central connecting helix. The three-dimensional structure of FtsZ is similar to the structure of alpha- and beta-tubulin.
Tubulin and FtsZ share a common fold of two domains connected by a central helix. Structure-based sequence alignment shows that common residues localize in the nucleotide-binding site and a region that interacts with the nucleotide of the next tubulin subunit in the protofilament, suggesting that tubulin and FtsZ use similar contacts to form filaments. Surfaces that would make lateral interactions between protofilaments or interact with motor proteins are, however, different. The highly conserved nucleotide-binding sites of tubulin and FtsZ clearly differ from those of EF-Tu and other GTPases, while resembling the nucleotide site of glyceraldehyde-3-phosphate dehydrogenase. Thus, tubulin and FtsZ form a distinct family of GTP-hydrolyzing proteins.
Bacterial cell division depends on the formation of a cytokinetic ring structure, the Z-ring. The bacterial tubulin homologue FtsZ is required for Z-ring formation. FtsZ assembles into various polymeric forms in vitro, indicating a structural role in the septum of bacteria. We have used recombinant FtsZ1 protein from M. jannaschii to produce helical tubes and sheets with high yield using the GTP analogue GMPCPP [guanylyl-(alpha,beta)-methylene-diphosphate]. The sheets appear identical to the previously reported Ca++-induced sheets of FtsZ from M. jannaschii that were shown to consist of 'thick'-filaments in which two protofilaments run in parallel. Tubes assembled either in Ca++ or in GMPCPP contain filaments whose dimensions indicate that they could be equivalent to the 'thick'-filaments in sheets. Some tubes are hollow but others are filled by additional protein density. Helical FtsZ tubes differ from eukaryotic microtubules in that the filaments curve around the filament axis with a pitch of approximately 430 A for Ca++-induced tubes or 590 - 620 A for GMPCPP. However, their assembly in vitro as well-ordered polymers over distances comparable to the inner circumference of a bacterium may indicate a role in vivo. Their size and stability make them suitable for use in motility assays.
FtsZ is the first protein recruited to the bacterial division site, where it forms the cytokinetic Z ring. We have determined the functional energetics of FtsZ assembly, employing FtsZ from the thermophilic Archaea Methanococcus jannaschii bound to GTP, GMPCPP, GDP, or GMPCP, under different solution conditions. FtsZ oligomerizes in a magnesium-insensitive manner. FtsZ cooperatively assembles with magnesium and GTP or GMPCPP into large polymers, following a nucleated condensation polymerization mechanism, under nucleotide hydrolyzing and non-hydrolyzing conditions. The effect of temperature on the critical concentration indicates polymer elongation with an apparent heat capacity change of -800 +/- 100 cal mol-1 K-1 and positive enthalpy and entropy changes, compatible with axial hydrophobic contacts of each FtsZ in the polymer, and predicts optimal polymer stability near 75 degrees C. Assembly entails the binding of one medium affinity magnesium ion and the uptake of one proton per FtsZ. Interestingly, GDP- or GMPCP-liganded FtsZ cooperatively form helically curved polymers, with an elongation only 1-2 kcal mol-1 more unfavorable than the straight polymers formed with nucleotide triphosphate, suggesting a physiological requirement for FtsZ polymerization inhibitors. This GTP hydrolysis switch should provide the basic properties for FtsZ polymer disassembly and its functional dynamics.
Microtubules can be assembled in vitro from purified alpha/beta tubulin heterodimers in the presence of GTP. Tubulin is routinely obtained from animal brain tissue through repetitive cycles of polymerization-depolymerization, followed by ion-exchange chromatography to remove any contaminating microtubule-associated proteins and motors. Here, we show that only two cycles of polymerization-depolymerization of pig brain tubulin in the presence of a high-molarity PIPES buffer allow the efficient removal of contaminating proteins and production of a high-concentration tubulin solution. The proposed protocol is rapid and yields more active tubulin than the traditional ion-exchange chromatography-based procedures.
The small-molecule topology generator PRODRG is described, which takes input from existing coordinates or various two-dimensional formats and automatically generates coordinates and molecular topologies suitable for X-ray refinement of protein-ligand complexes. Test results are described for automatic generation of topologies followed by energy minimization for a subset of compounds from the Cambridge Structural Database, which shows that, within the limits of the empirical GROMOS87 force field used, structures with good geometries are generated. X-ray refinement in X-PLOR/CNS, REFMAC and SHELX using PRODRG-generated topologies produces results comparable to refinement with topologies from the standard libraries. However, tests with distorted starting coordinates show that PRODRG topologies perform better, both in terms of ligand geometry and of crystallographic R factors.
The prokaryotic tubulin homolog FtsZ polymerizes into a ring structure essential for bacterial cell division. We have used refolded FtsZ to crystallize a tubulin-like protofilament. The N- and C-terminal domains of two consecutive subunits in the filament assemble to form the GTPase site, with the C-terminal domain providing water-polarizing residues. A domain-swapped structure of FtsZ and biochemical data on purified N- and C-terminal domains show that they are independent. This leads to a model of how FtsZ and tubulin polymerization evolved by fusing two domains. In polymerized tubulin, the nucleotide-binding pocket is occluded, which leads to nucleotide exchange being the rate-limiting step and to dynamic instability. In our FtsZ filament structure the nucleotide is exchangeable, explaining why, in this filament, nucleotide hydrolysis is the rate-limiting step during FtsZ polymerization. Furthermore, crystal structures of FtsZ in different nucleotide states reveal notably few differences.
Screening of 120 taxanes identified a number of compounds that exhibited significant antituberculosis activity. Rational optimization of selected compounds led to the discovery that the C-seco-taxane-multidrug-resistance (MDR) reversal agents (C-seco-TRAs) are noncytotoxic at the upper limit of solubility and detection (>80 microM), while maintaining MIC(99) values of 1.25-2.5 microM against drug-resistant and drug-sensitive strains of Mycobacterium tuberculosis (MTB). Treatment of MTB cells with TRA 3aa and 10a at the MIC caused filamentation and prolongation of the cells, a phenotypic response to FtsZ inactivation.
Bacterial cells contain a variety of structural filamentous proteins necessary for the spatial regulation of cell shape, cell division, and chromosome segregation, analogous to the eukaryotic cytoskeletal proteins. The molecular mechanisms by which these proteins function are beginning to be revealed, and these proteins show numerous three-dimensional structural features and biochemical properties similar to those of eukaryotic actin and tubulin, revealing their evolutionary relationship. Recent technological advances have illuminated links between cell division and chromosome segregation, suggesting a higher complexity and organization of the bacterial cell than was previously thought.
The emergence of multi-drug resistant Mycobacterium tuberculosis (Mtb) strains has made many of the currently available anti-TB drugs ineffective. Accordingly there is a pressing need to identify new drug targets. FtsZ, a bacterial tubulin homologue, is an essential cell division protein that polymerizes in a GTP-dependent manner, forming a highly dynamic cytokinetic ring, designated as the Z ring, at the septum site. Following recruitment of other cell division proteins, the Z ring contracts, resulting in closure of the septum and then formation of two daughter cells. Since inactivation of FtsZ or alteration of FtsZ assembly results in the inhibition of Z ring and septum formation, FtsZ is a very promising target for new antimicrobial drug development. This review describes the function and dynamic behaviors of FtsZ, its homology to tubulin, and recent development of FtsZ inhibitors as potential anti-TB agents.
Prokaryotic cell division protein FtsZ, an assembling GTPase, directs the formation of the septosome between daughter cells. FtsZ is an attractive target for the development of new antibiotics. Assembly dynamics of FtsZ is regulated by the binding, hydrolysis, and exchange of GTP. We have determined the energetics of nucleotide binding to model apoFtsZ from Methanococcus jannaschii and studied the kinetics of 2'/3'-O-(N-methylanthraniloyl) (mant)-nucleotide binding and dissociation from FtsZ polymers, employing calorimetric, fluorescence, and stopped-flow methods. FtsZ binds GTP and GDP with K(b) values ranging from 20 to 300 microm(-1) under various conditions. GTP.Mg(2+) and GDP.Mg(2+) bind with slightly reduced affinity. Bound GTP and the coordinated Mg(2+) ion play a minor structural role in FtsZ monomers, but Mg(2+)-assisted GTP hydrolysis triggers polymer disassembly. Mant-GTP binds and dissociates quickly from FtsZ monomers, with approximately 10-fold lower affinity than GTP. Mant-GTP displacement measured by fluorescence anisotropy provides a method to test the binding of any competing molecules to the FtsZ nucleotide site. Mant-GTP is very slowly hydrolyzed and remains exchangeable in FtsZ polymers, but it becomes kinetically stabilized, with a 30-fold slower k(+) and approximately 500-fold slower k(-) than in monomers. The mant-GTP dissociation rate from FtsZ polymers is comparable with the GTP hydrolysis turnover and with the reported subunit turnover in Escherichia coli FtsZ polymers. Although FtsZ polymers can exchange nucleotide, unlike its eukaryotic structural homologue tubulin, GDP dissociation may be slow enough for polymer disassembly to take place first, resulting in FtsZ polymers cycling with GTP hydrolysis similarly to microtubules.
The capacity of the centrosome to influence the lattice structure of nucleated microtubules was studied in vitro. Brain microtubules self-assembled to give predominantly (98%) 14-protofilament microtubules. However, under exactly the same conditions of assembly they grew off of purified centrosomes from neuroblastoma cells to give mostly (82%) 13- protofilament microtubules. Thus, the nucleation sites on the centrosome constrained the microtubule lattice to yield the number of protofilaments usually found in vivo.
The reaction between 1-B-D-arabinofuranosylcytosine hydrochloride (araC.HCl) and a slight molar excess of m-chloroperbenzoic acid (MCPBA) in an aprotic solvent such as dimethylformamide (DMF), dimethylacetamide (DMA), or hexamethylphosphorus triaide was examined. Chromatographic evaluation of the reaction mixture showed two major components, unreacted starting material, and a reaction product having a slightly greater R/sub f/ value. The reaction between MCPBA and other pyrimidine and purine derivatives in dipolar aprotic solvents containing HCl was investigated. In the pyrimidine series both uracil and cytosine derivatives gave the corresponding 5-chloro derivatives in high yield after a facile workup. Application of the same reaction conditions to the purine derivatives adenosine and guanosine gave the 8-chloro nucleosides in good yield, thus producing 8-chlorogyanosine. The product 9-B-D-arabinofuranosyl-8-chloroadenine was obtained when the reaction was applied to the potent antiviral agent 9-B-D-arabinofuraosyladenine. The new chlorination method gives good to excellent yields, short reaction times, mild reaction conditions and ready availability of starting materials. 1 table. (DP)
The cell division protein FtsZ is a GTPase structurally related to tubulin and, like tubulin, it assembles in vitro into filaments, sheets and other structures. To study the roles that GTP binding and hydrolysis play in the dynamics of FtsZ polymerization, the nucleotide contents of FtsZ were measured under different polymerizing conditions using a nitrocellulose filter-binding assay, whereas polymerization of the protein was followed in parallel by light scattering. Unpolymerized FtsZ bound 1 mol of GTP mol−1 protein monomer. At pH 7.5 and in the presence of Mg2+ and K+, there was a strong GTPase activity; most of the bound nucleotide was GTP during the first few minutes but, later, the amount of GTP decreased in parallel with depolymerization, whereas the total nucleotide contents remained invariant. These results show that the long FtsZ polymers formed in solution contain mostly GTP. Incorporation of nucleotides into the protein was very fast either when the label was introduced at the onset of the reaction or subsequently during polymerization. Molecular modelling of an FtsZ dimer showed the presence of a cleft between the two subunits maintaining the nucleotide binding site open to the medium. These results show that the FtsZ polymers are highly dynamic structures that quickly exchange the bound nucleotide, and this exchange can occur in all the subunits.
Guanine, guanosine, and 5′-guanylic acid were methylated with t-butylhydroperoxide in the presence of ferrous ion to give the corresponding 8-Me derivatives in fairly good yields. Adenine, hypoxanthine, and their ribosides also underwent the methylation to give the corresponding mono- and di-methyl derivatives where Me groups were substituted in position-2, -8, or both. N,N-Dimethylcarbamoyl group was introduced to position-8 of guanosine under a similar condition using dimethylformamide as the reaction solvent.
FtsZ assembles in vitro into protofilaments that can adopt two conformations—the straight conformation, which can assemble
further into two-dimensional protofilament sheets, and the curved conformation, which forms minirings about 23 nm in diameter.
Here, we describe the structure of FtsZ tubes, which are a variation of the curved conformation. In the tube the curved protofilament
forms a shallow helix with a diameter of 23 nm and a pitch of 18 or 24°. We suggest that this shallow helix is the relaxed
structure of the curved protofilament in solution. We provide evidence that GTP favors the straight conformation while GDP
favors the curved conformation. In particular, exclusively straight protofilaments and protofilament sheets are assembled
in GMPCPP, a nonhydrolyzable GTP analog, or in GTP following chelation of Mg, which blocks GTP hydrolysis. Assembly in GDP
produces exclusively tubes. The transition from straight protofilaments to the curved conformation may provide a mechanism
whereby the energy of GTP hydrolysis is used to generate force for the constriction of the FtsZ ring in cell division.
The question of whether nonhydrolyzable nucleotide analogues and other nucleoside triphosphates support tubulin assembly was addressed. Tubulin which contained residual GTP at the exchangeable site polymerized in the absence of added GTP in the presence of DMSO or glycerol. After maximum absorbance was reached, disassembly occurred at a slow rate. When 0.5 mM GMPPCP, GMPPNP, or ATP was included in the assembly reaction, disassembly did not occur, and about 0.1 mol of these nucleotides per mole of tubulin was incorporated into the protein. When 5 mM nucleotide was used or alkaline phosphatase was included in the case of the nonhydrolyzable analogues, a greater amount of assembly occurred and about 0.7-0.8 mol of analogue was incorporated. The products of the assembly reaction were cold-labile microtubules and protofilament ribbons. After cold-depolymerization of the microtubules and ribbons, a second cycle of assembly produced some microtubules, but cold-stable amorphous polymers were the major product. In addition, when GTP at the exchangeable site was first removed by a cycle of assembly, followed by depolymerization, assembly in the presence of GMPPCP, GMPPNP, or ATP produced a mixture of microtubules and cold-stable polymers, both of which contained bound analogue. Incorporation of GMPPCP, GMPPNP, or ATP into polymerized tubulin always occurred at the expense of GDP at the exchangeable site, the content of which decreased correspondingly. Incubation of tubulin with 5 mM GMPPCP, GMPPNP, or ATP under nonassembly conditions also displaced GDP.(ABSTRACT TRUNCATED AT 250 WORDS)
Four analogues of guanosine 5'-triphosphate (GTP) (dGTP, 3'-deoxy-GTP, arabinosyl-GTP, and 2',3'-dideoxy-GTP), which support more rapid and extensive microtubule assembly than GTP, were hydrolyzed more rapidly than GTP in reaction mixtures containing tubulin plus microtubule-associated proteins (MAPs). As with GTP, hydrolysis of the four analogues was initially closely coupled to the onset of polymerization and continued at a slower rate at the turbidity plateau. Relative to GTP, however, these analogues (and the cognate GDP analogues), particularly 3'-deoxy-GTP and 2',3'-dideoxy-GTP, bound poorly to tubulin and had a reduced ability to displace bound radiolabeled GDP under nonpolymerizing reaction conditions. Despite their reduced binding to the tubulin dimer, if polymerization occurred, all four analogues were incorporated into microtubules (as the diphosphates) in stoichiometric amounts comparable to the incorporation of GTP (in the form of GDP) with displacement of the GDP initially present in the exchangeable site. Microtubule nucleation was specifically enhanced in the presence of the analogues. With MAPs the analogues initiated microtubule assembly at temperatures 10-15 degrees C below that required by the GTP-supported reaction, and the average microtubule length was significantly reduced. In addition, MAP-independent polymerization occurred only with 2',3'-dideoxy-GTP with tubulin at 1.0 mg/mL, with the other three analogues at 2.0 mg/mL, and with GTP at 5.0 mg/mL. GTP inhibited analogue-supported polymerization at 20 degrees C with MAPs and at 37 degrees C without MAPs (tubulin, 3.5 mg/mL). Both 3'-deoxy-GTP and 2',3'-dideoxy-GTP were poor inhibitors of GTP binding and hydrolysis, but GTP potently inhibited the more vigorous hydrolysis of these analogues. We conclude that alteration of the ribose moiety reduces the affinity of a guanine nucleotide for the exchangeable site of tubulin but that a nucleotide's affinity for this site is not the major factor in its ability to support the nucleation of tubulin polymerization.
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FtsZ forms a cytokinetic ring, designated the Z ring, that directs cytokinesis in prokaryotes. It has limited sequence similarity to eukaryotic tubulins and, like tubulin, it has GTPase activity and the ability to assemble into various structures including protofilaments, bundles and minirings. By using both electron microscopy and sedimentation, we demonstrate that FtsZ from Escherichia coli undergoes a strictly GTP-dependent polymerization and the polymers disappear as the GTP is consumed. Thus, FtsZ polymerization, like that of tubulin, is dynamic and regulated by GTP hydrolysis. These results provide the basis for the dynamics of the Z ring and favor a model in which the Z ring is formed by a nucleation event.
We synthesized 27 GTP analogues with modification or substitution at positions C2, C6, C8 and ribose moiety to investigate their effect on microtubule (Mt) assembly. It was found that C2 and C6 are both functional for the analogues supporting Mt assembly. It was surprising to find that 2-amino- ATP (n2ATP) substantially supports assembly, and that the appearance of the assembled Mts was indistinguishable from those assembled in the standard GTP assembly buffer solution. Furthermore, 2-amino dATP and dGTP are even more potent than GTP in supporting assembly. The substitution of oxo group at C6 with reactive thiol largely reduced the activity of the analogue to support assembly. When free rotation of the glycosidic linkage of GTP was blocked by the introduction of sulfur atom between C8 and C2' of ribose moiety, it resulted in total suppression of assembly. Purine nucleoside triphosphate was found to support assembly better than GTP, and even more efficient was 2-amino purine nucleoside triphosphate. Interestingly, their deoxy-type analogues were totally inhibitory. Although 2-amino 8-hydroxy ATP and other analogues supported assembly much better than did GTP, their diphosphate analogues were totally incapable of supporting assembly. Finally, bulky fluorescent probes were introduced at C3' of ribose moiety (Mant-8-Br-GTP or Mant-GTP) to visualize the fluorescent signal in assembled Mts. Even in this case, the number of most protofilaments was found to be 14, consistent with that found in Mts assembled in GTP standard buffer solution.
FtsZ, a tubulin homologue, forms a cytokinetic ring at the site of cell division in prokaryotes. The ring is thought to consist of polymers that assemble in a strictly GTP-dependent way. GTP, but not guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S), has been shown to induce polymerization of FtsZ, whereas in vitro Ca2+ is known to inhibit the GTP hydrolysis activity of FtsZ. We have studied FtsZ dynamics at limiting GTP concentrations in the presence of 10 mM Ca2+. GTP and its non-hydrolysable analogue GTP-gamma-S bind FtsZ with similar affinity, whereas the non-hydrolysable analogue guanylyl-imidodiphosphate (GMP-PNP) is a poor substrate. Preformed FtsZ polymers can be stabilized by GTP-gamma-S and are destabilized by GDP. As more than 95% of the nucleotide associated with the FtsZ polymer is in the GDP form, it is concluded that GTP hydrolysis by itself does not trigger FtsZ polymer disassembly. Strikingly, GTP-gamma-S exchanges only a small portion of the FtsZ polymer-bound GDP. These data suggest that FtsZ polymers are stabilized by a small fraction of GTP-containing FtsZ subunits. These subunits may be located either throughout the polymer or at the polymer ends, forming a GTP cap similar to tubulin.
The cell division protein FtsZ is a GTPase structurally related to tubulin and, like tubulin, it assembles in vitro into filaments, sheets and other structures. To study the roles that GTP binding and hydrolysis play in the dynamics of FtsZ polymerization, the nucleotide contents of FtsZ were measured under different polymerizing conditions using a nitrocellulose filter-binding assay, whereas polymerization of the protein was followed in parallel by light scattering. Unpolymerized FtsZ bound 1 mol of GTP mol(-1) protein monomer. At pH 7.5 and in the presence of Mg(2+) and K(+), there was a strong GTPase activity; most of the bound nucleotide was GTP during the first few minutes but, later, the amount of GTP decreased in parallel with depolymerization, whereas the total nucleotide contents remained invariant. These results show that the long FtsZ polymers formed in solution contain mostly GTP. Incorporation of nucleotides into the protein was very fast either when the label was introduced at the onset of the reaction or subsequently during polymerization. Molecular modelling of an FtsZ dimer showed the presence of a cleft between the two subunits maintaining the nucleotide binding site open to the medium. These results show that the FtsZ polymers are highly dynamic structures that quickly exchange the bound nucleotide, and this exchange can occur in all the subunits.
The essential prokaryotic cell division protein FtsZ is a tubulin homologue that forms a ring at the division site. FtsZ forms polymers in a GTP-dependent manner. Recent biochemical evidence has shown that FtsZ forms multimeric structures in vitro and in vivo and functions as a self-activating GTPase. Structural analysis of FtsZ points to an important role for the highly conserved tubulin-like loop 7 (T7-loop) in the self-activation of GTP hydrolysis. The T7-loop was postulated to form the active site together with the nucleotide-binding site on an adjacent FtsZ monomer. To characterize the role of the T7-loop of Escherichia coli FtsZ, we have mutagenized residues M206, N207, D209, D212, and R214. All the mutant proteins, except the R214 mutant, are severely affected in polymerization and GTP hydrolysis. Charged residues D209 and D212 cannot be substituted with a glutamate residue. All mutants interact with wild-type FtsZ in vitro, indicating that the T7-loop mutations do not abolish FtsZ self-association. Strikingly, in mixtures of wild-type and mutant proteins, most mutants are capable of inhibiting wild-type GTP hydrolysis. We conclude that the T7-loop is part of the active site for GTP hydrolysis, formed by the association of two FtsZ monomers.
Modification of nucleosides to give pharmaceutically active compounds, mutagenesis models, and oligonucleotide structural probes continues to be of great interest. The aqueous-phase modification of unprotected halonucleosides is reported herein. Using a catalyst derived from tris(3-sulfonatophenyl)phosphine (TPPTS) and palladium acetate, 8-bromo-2'-deoxyguanosine (8-BrdG) is coupled with arylboronic acids to give 8-aryl-2'-deoxyguanosine adducts (8-ArdG) in excellent yield in a 2:1 water:acetonitrile solvent mixture. The TPPTS ligand was found to be superior to water-soluble alkylphosphines for this coupling reaction. The coupling chemistry has been extended to 8-bromo-2'-deoxyadenosine (8-BrdA) and 5-iodo-2'-deoxyuridine (5-IdU), as well as the ribonucleosides 8-bromoguanosine and 8-bromoadenosine. Good to excellent yields of arylated adducts are obtained in all cases. With use of tri(4,6-dimethyl-3-sulfonatophenyl)phosphine (TXPTS), the Suzuki coupling of 8-BrdA and 5-IdU can be accomplished in less than 1 h at room temperature. This methodology represents an efficient and general method for halonucleoside arylation that does not require prior protection of the nucleoside.
FtsZ is a prokaryotic tubulin homologue that polymerizes into a dynamic ring during cell division. GTP binding and hydrolysis provide the energy for FtsZ dynamics. However, the precise role of hydrolysis in polymer assembly and turnover is not understood, limiting our understanding of how FtsZ functions in the cell. Here we investigate GTP hydrolysis during the FtsZ polymerization cycle using several complementary approaches that avoid technical caveats of previous studies. We find that at steady state approximately 80% of FtsZ polymer subunits are bound to GTP. In addition, we use pre-steady-state, single turnover assays to directly measure the rate of hydrolysis. Hydrolysis was found to occur at approximately 8/min and to be a rate-limiting step in GTP turnover; phosphate release rapidly followed. These results clarify previously conflicting results in the literature and suggest that pure FtsZ polymers, unlike microtubules, may not be able to undergo dynamic instability or to store energy in the polymer for force production.
This minireview summarizes the syntheses of various purinenucleotide analogues and their effects on microtubule (Mt) assembly. 27 analogues were so far synthesized and, together with 3 analogues commercially available (ITP, XTP and dGTP), their effects on Microtubule assembly were investigated. The positions C2, C6, C8, and ribose moiety of purine nucleotides were modified or substituted. It was found that the microenvironments of the purine base and ribose moiety are important for the nucleotides to support Mt assembly. Introduction of amino group into position C2 of ATP, formation of 2-amino ATP, caused Mt assembly substantially. 2-Amino deoxy ATP and deoxy GTP are more potent than GTP in supporting assembly. The introduction of reactive thiol group into C6 (6-SH-GTP) largely reduces the activity of the analogue to support assembly. However, sequestering reactivity of the thiol group by association with methyl group largely recovers the ability of the analogue to promote assembly. Free rotation of the glycosidic linkage was found to be also innevitable in promoting assembly, as the introduction of sulfur atom between C8 of the purine base and C2' of the ribose moiety (formation of 8,2'-S-cyclo purine nucleotides) caused total inhibition. Purinenucleoside triphosphate supports assembly better than GTP but the deoxy-type analogues are totally inhibitory. 2-Amino-8-hydroxy ATP and other analogues support assembly much better than does GTP. However, their diphosphate analogues are totally incapable of supporting assembly. Introduction of a bulky fluorescent probes into C3' can be made to visualize the fluorescent signal in assembled Mts. Together with the suggestions proposed from electron chrystallography of zinc-induced tubulin sheets, interactions of the purine base and ribose moieties with surrounding amino acid residues are discussed.
This paper reviews the mathematical basis of maximum likelihood. The likelihood function for macromolecular structures is extended to include prior phase information and experimental standard uncertainties. The assumption that different parts of a structure might have different errors is considered. A method for estimating sigma(A) using 'free' reflections is described and its effects analysed. The derived equations have been implemented in the program REFMAC. This has been tested on several proteins at different stages of refinement (bacterial alpha-amylase, cytochrome c', cross-linked insulin and oligopeptide binding protein). The results derived using the maximum-likelihood residual are consistently better than those obtained from least-squares refinement.
The prokaryotic tubulin homologue FtsZ plays a key role in bacterial cell division. Selective inhibitors of the GTP-dependent polymerization of FtsZ are expected to result in a new class of antibacterial agents. One of the challenges is to identify compounds which do not affect the function of tubulin and various other GTPases in eukaryotic cells. We have designed a novel inhibitor of FtsZ polymerization based on the structure of the natural substrate GTP. The inhibitory activity of 8-bromoguanosine 5'-triphosphate (BrGTP) was characterized by a coupled assay, which allows simultaneous detection of the extent of polymerization (via light scattering) and GTPase activity (via release of inorganic phosphate). We found that BrGTP acts as a competitive inhibitor of both FtsZ polymerization and GTPase activity with a Ki for GTPase activity of 31.8 +/- 4.1 microM. The observation that BrGTP seems not to inhibit tubulin assembly suggests a structural difference of the GTP-binding pockets of FtsZ and tubulin.
Microtubules are cytoskeletal polymers made of repeating alphabeta-tubulin heterodimers that play essential roles in all eukaryotic cells. The complex dynamic behavior of microtubules, which is ultimately due to the tubulin subunit structure and its intrinsic GTPase activity, is key to the functions of these ubiquitous polymers. Microtubule assembly and disassembly do not take place by simple helical growth and shrinkage via individual subunits, but rather involve transient polymer intermediates, distinct from the microtubule, without parallel in other biological self-assembly systems. The discovery of these intermediates a decade ago has been followed recently by quantitative descriptions of their structure and their relationship to nucleotide state.