RESUMO
Treadmilling protein filaments perform essential cellular functions by growing from one end while shrinking from the other, driven by nucleotide hydrolysis. Bacterial cell division relies on the primitive tubulin homolog FtsZ, a target for antibiotic discovery that assembles into single treadmilling filaments that hydrolyse GTP at an active site formed upon subunit association. We determined high-resolution filament structures of FtsZ from the pathogen Staphylococcus aureus in complex with different nucleotide analogs and cations, including mimetics of the ground and transition states of catalysis. Together with mutational and biochemical analyses, our structures reveal interactions made by the GTP γ-phosphate and Mg2+ at the subunit interface, a K+ ion stabilizing loop T7 for co-catalysis, new roles of key residues at the active site and a nearby crosstalk area, and rearrangements of a dynamic water shell bridging adjacent subunits upon GTP hydrolysis. We propose a mechanistic model that integrates nucleotide hydrolysis signaling with assembly-associated conformational changes and filament treadmilling. Equivalent assembly mechanisms may apply to more complex tubulin and actin cytomotive filaments that share analogous features with FtsZ.
Assuntos
Proteínas do Citoesqueleto , Nucleotídeos , Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/metabolismo , Guanosina Trifosfato/metabolismo , Tubulina (Proteína)RESUMO
Cell division in most bacteria is directed by FtsZ, a conserved tubulin-like GTPase that assembles forming the cytokinetic Z-ring and constitutes a target for the discovery of new antibiotics. The developmental regulator MciZ, a 40-amino acid peptide endogenously produced during Bacillus subtilis sporulation, halts cytokinesis in the mother cell by inhibiting FtsZ. The crystal structure of a FtsZ:MciZ complex revealed that bound MciZ extends the C-terminal ß-sheet of FtsZ blocking its assembly interface. Here we demonstrate that exogenously added MciZ specifically inhibits B. subtilis cell division, sporulation and germination, and provide insight into MciZ molecular recognition by FtsZ from different bacteria. MciZ and FtsZ form a complex with sub-micromolar affinity, analyzed by analytical ultracentrifugation, laser biolayer interferometry and isothermal titration calorimetry. Synthetic MciZ analogs, carrying single amino acid substitutions impairing MciZ ß-strand formation or hydrogen bonding to FtsZ, show a gradual reduction in affinity that resembles their impaired activity in bacteria. Gene sequences encoding MciZ spread across genus Bacillus and synthetic MciZ slows down cell division in Bacillus species, including pathogenic Bacillus cereus and Bacillus anthracis. Moreover, B. subtilis MciZ is recognized by the homologous FtsZ from Staphylococcus aureus and inhibits division when it is expressed into S. aureus cells.
Assuntos
Bacillus subtilis/efeitos dos fármacos , Proteínas de Bactérias/antagonistas & inibidores , Divisão Celular/efeitos dos fármacos , Proteínas do Citoesqueleto/antagonistas & inibidores , Peptídeos/farmacologia , Substituição de Aminoácidos , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Sítios de Ligação , Proteínas do Citoesqueleto/genética , Regulação Bacteriana da Expressão Gênica , Peptídeos/síntese química , Ligação Proteica , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genéticaRESUMO
The marine natural product zampanolide and analogues thereof constitute a new chemotype of taxoid site microtubule-stabilizing agents with a covalent mechanism of action. Zampanolide-ligated tubulin has the switch-activation loop (M-loop) in the assembly prone form and, thus, represents an assembly activated state of the protein. In this study, we have characterized the biochemical properties of the covalently modified, activated tubulin dimer, and we have determined the effect of zampanolide on tubulin association and the binding of tubulin ligands at other binding sites. Tubulin activation by zampanolide does not affect its longitudinal oligomerization but does alter its lateral association properties. The covalent binding of zampanolide to ß-tubulin affects both the colchicine site, causing a change of the quantum yield of the bound ligand, and the exchangeable nucleotide binding site, reducing the affinity for the nucleotide. While these global effects do not change the binding affinity of 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC) (a reversible binder of the colchicine site), the binding affinity of a fluorescent analogue of GTP (Mant-GTP) at the nucleotide E-site is reduced from 12 ± 2 × 105 M-1 in the case of unmodified tubulin to 1.4 ± 0.3 × 105 M-1 in the case of the zampanolide tubulin adduct, indicating signal transmission between the taxane site and the colchicine and nucleotide sites of ß-tubulin.
Assuntos
Sítios de Ligação/fisiologia , Hidrocarbonetos Aromáticos com Pontes/metabolismo , Colchicina/metabolismo , Macrolídeos/metabolismo , Nucleotídeos/metabolismo , Taxoides/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Produtos Biológicos/metabolismo , Bovinos , Humanos , Ligantes , Microtúbulos/metabolismoRESUMO
FtsZ is a self-assembling GTPase that forms, below the inner membrane, the mid-cell Z-ring guiding bacterial division. FtsZ monomers polymerize head to tail forming tubulin-like dynamic protofilaments, whose organization in the Z-ring is an unresolved problem. Rather than forming a well-defined structure, FtsZ protofilaments laterally associate in vitro into polymorphic condensates typically imaged on surfaces. We describe here nanoscale self-organizing properties of FtsZ assemblies in solution that underlie Z-ring assembly, employing time-resolved x-ray scattering and cryo-electron microscopy. We find that FtsZ forms bundles made of loosely bound filaments of variable length and curvature. Individual FtsZ protofilaments further bend upon nucleotide hydrolysis, highlighted by the observation of some large circular structures with 2.5-5° curvature angles between subunits, followed by disassembly end-products consisting of highly curved oligomers and 16-subunit -220 Å diameter mini-rings, here observed by cryo-electron microscopy. Neighbor FtsZ filaments in bundles are laterally spaced 70 Å, leaving a gap in between. In contrast, close contact between filament core structures (â¼50 Å spacing) is observed in straight polymers of FtsZ constructs lacking the C-terminal tail, which is known to provide a flexible tether essential for FtsZ functions in cell division. Changing the length of the intrinsically disordered C-tail linker modifies the interfilament spacing. We propose that the linker prevents dynamic FtsZ protofilaments in bundles from sticking to one another, holding them apart at a distance similar to the lateral spacing observed by electron cryotomography in several bacteria and liposomes. According to this model, weak interactions between curved polar FtsZ protofilaments through their the C-tails may facilitate the coherent treadmilling dynamics of membrane-associated FtsZ bundles in reconstituted systems, as well as the recently discovered movement of FtsZ clusters around bacterial Z-rings that is powered by GTP hydrolysis and guides correct septal cell wall synthesis and cell division.
Assuntos
Proteínas Arqueais/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas Arqueais/química , Bacillus subtilis , Proteínas de Bactérias/química , Microscopia Crioeletrônica , Proteínas do Citoesqueleto/química , Escherichia coli , Hidrólise , Methanocaldococcus , Modelos Moleculares , Polímeros , Domínios Proteicos , Multimerização Proteica , Espalhamento a Baixo Ângulo , Soluções/química , Difração de Raios XRESUMO
The recent success of antibody-drug conjugates (ADCs) in the treatment of cancer has led to a revived interest in microtubule-destabilizing agents. Here, we determined the high-resolution crystal structure of the complex between tubulin and maytansine, which is part of an ADC that is approved by the US Food and Drug Administration (FDA) for the treatment of advanced breast cancer. We found that the drug binds to a site on ß-tubulin that is distinct from the vinca domain and that blocks the formation of longitudinal tubulin interactions in microtubules. We also solved crystal structures of tubulin in complex with both a variant of rhizoxin and the phase 1 drug PM060184. Consistent with biochemical and mutagenesis data, we found that the two compounds bound to the same site as maytansine and that the structures revealed a common pharmacophore for the three ligands. Our results delineate a distinct molecular mechanism of action for the inhibition of microtubule assembly by clinically relevant agents. They further provide a structural basis for the rational design of potent microtubule-destabilizing agents, thus opening opportunities for the development of next-generation ADCs for the treatment of cancer.
Assuntos
Antibióticos Antineoplásicos/química , Antineoplásicos Fitogênicos/química , Macrolídeos/química , Maitansina/química , Microtúbulos/química , Policetídeos/química , Pironas/química , Moduladores de Tubulina/química , Tubulina (Proteína)/química , Animais , Antibióticos Antineoplásicos/uso terapêutico , Antineoplásicos Fitogênicos/uso terapêutico , Sítios de Ligação , Neoplasias da Mama/tratamento farmacológico , Bovinos , Ensaios Clínicos Fase I como Assunto , Cristalografia por Raios X , Feminino , Humanos , Macrolídeos/uso terapêutico , Maitansina/uso terapêutico , Policetídeos/uso terapêutico , Pironas/uso terapêutico , Moduladores de Tubulina/uso terapêuticoRESUMO
The authors of this study evaluated the factor structure, internal consistency, and concurrent and discriminant validity of the Posttraumatic Cognitions Inventory (PTCI; Foa et al. 1999) in a sample of 107 female survivors of sexual assault with a mean age of 29.1 years (SD = 7.7). All participants were recruited between July 2010 and December 2014 from a care center for sexual assault victims in Madrid, Spain. Results supported the three-factor structure of the PTCI: (1) negative cognition about self, (2) negative cognition about the world, and (3) self-blame. The negative cognitions about self and the world subscales showed adequate internal consistency (Cronbach's alpha = 0.92 and 0.82, respectively), as well as good concurrent and discriminant validity. Nevertheless, the subscale measuring self-blame showed poor internal consistency (Cronbach's alpha = 0.75) and discriminant validity. The PTCI presents sound psychometric characteristics and has the potential to contribute to women's sexual posttrauma assessment.
Assuntos
Autoimagem , Delitos Sexuais/psicologia , Transtornos de Estresse Pós-Traumáticos/psicologia , Sobreviventes/psicologia , Adulto , Feminino , Humanos , Psicometria , Reprodutibilidade dos Testes , Adulto JovemRESUMO
Partition systems are responsible for the process whereby large and essential plasmids are accurately positioned to daughter cells during bacterial division. They are typically made of three components: a centromere-like DNA zone, an adaptor protein, and an assembling protein that is either a Walker-box ATPase (type I) or an actin-like ATPase (type II). A recently described type III segregation system has a tubulin/FtsZ-like protein, called TubZ, for plasmid movement. Here, we present the 2.3 Å structure and dynamic assembly of a TubZ tubulin homolog from a bacteriophage and unravel the Clostridium botulinum phage c-st type III partition system. Using biochemical and biophysical approaches, we prove that a gene upstream from tubZ encodes the partner TubR and localize the centromeric region (tubS), both of which are essential for anchoring phage DNA to the motile TubZ filaments. Finally, we describe a conserved fourth component, TubY, which modulates the TubZ-R-S complex interaction.
Assuntos
Bacteriófagos/química , Clostridium botulinum/fisiologia , Citocinese/fisiologia , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Plasmídeos/metabolismo , Tubulina (Proteína)/química , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Sequência de Bases , Biofísica , Centrômero/metabolismo , Clonagem Molecular , Análise por Conglomerados , Biologia Computacional , Cristalização , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNA , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
Proteins involved in bacterial cell division often do not have a counterpart in eukaryotic cells and they are essential for the survival of the bacteria. The genetic accessibility of many bacterial species in combination with the Green Fluorescence Protein revolution to study localization of proteins and the availability of crystal structures has increased our knowledge on bacterial cell division considerably in this century. Consequently, bacterial cell division proteins are more and more recognized as potential new antibiotic targets. An international effort to find small molecules that inhibit the cell division initiating protein FtsZ has yielded many compounds of which some are promising as leads for preclinical use. The essential transglycosylase activity of peptidoglycan synthases has recently become accessible to inhibitor screening. Enzymatic assays for and structural information on essential integral membrane proteins such as MraY and FtsW involved in lipid II (the peptidoglycan building block precursor) biosynthesis have put these proteins on the list of potential new targets. This review summarises and discusses the results and approaches to the development of lead compounds that inhibit bacterial cell division.
Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Proteínas de Bactérias/metabolismo , Divisão Celular , Parede Celular/metabolismo , Animais , Bactérias/metabolismo , HumanosRESUMO
FtsZ is the key protein of bacterial cell-division and target for new antibiotics. Selective inhibition of FtsZ polymerization without impairing the assembly of the eukaryotic homologue tubulin was demonstrated with C8-substituted guanine nucleotides. By combining NMR techniques with biochemical and molecular modeling procedures, we have investigated the molecular recognition of C8-substituted-nucleotides by FtsZ from Methanococcus jannaschii (Mj-FtsZ) and Bacillus subtilis (Bs-FtsZ). STD epitope mapping and trNOESY bioactive conformation analysis of each nucleotide were employed to deduce differences in their recognition mode by each FtsZ species. GMP binds in the same anti conformation as GTP, whereas 8-pyrrolidino-GMP binds in the syn conformation. However, the anti conformation of 8-morpholino-GMP is selected by Bs-FtsZ, while Mj-FtsZ binds both anti- and syn-geometries. The inhibitory potencies of the C8-modified-nucleotides on the assembly of Bs-FtsZ, but not of Mj-FtsZ, correlate with their binding affinities. Thus, MorphGTP behaves as a nonhydrolyzable analog whose binding induces formation of Mj-FtsZ curved filaments, resembling polymers formed by the inactive forms of this protein. NMR data, combined with molecular modeling protocols, permit explanation of the mechanism of FtsZ assembly impairment by C8-substituted GTP analogs. The presence of the C8-substituent induces electrostatic remodeling and small structural displacements at the association interface between FtsZ monomers to form filaments, leading to complete assembly inhibition or to formation of abnormal FtsZ polymers. The inhibition of bacterial Bs-FtsZ assembly may be simply explained by steric clashes of the C8-GTP-analogs with the incoming FtsZ monomer. This information may facilitate the design of antibacterial FtsZ inhibitors replacing GTP.
Assuntos
Bacillus subtilis/química , Proteínas de Bactérias/química , Proteínas do Citoesqueleto/química , Inibidores de Dissociação do Nucleotídeo Guanina/química , Methanocaldococcus/química , Ressonância Magnética Nuclear Biomolecular , Modelos Moleculares , Conformação de Ácido NucleicoRESUMO
The bacterial cell division protein FtsZ polymerizes in a GTP-dependent manner to form a Z-ring that marks the plane of division. As a validated antimicrobial target, considerable efforts have been devoted to identify small molecule FtsZ inhibitors. We recently discovered the chrysophaentins, a novel suite of marine natural products that inhibit FtsZ activity in vitro. These natural products along with a synthetic hemi-chrysophaentin exhibit strong antimicrobial activity toward a broad spectrum of Gram-positive pathogens. To define their mechanisms of FtsZ inhibition and determine their in vivo effects in live bacteria, we used GTPase assays and fluorescence anisotropy to show that hemi-chrysophaentin competitively inhibits FtsZ activity. Furthermore, we developed a model system using a permeable Escherichia coli strain, envA1, together with an inducible FtsZ-yellow fluorescent protein construct to show by fluorescence microscopy that both chrysophaentin A and hemi-chrysophaentin disrupt Z-rings in live bacteria. We tested the E. coli system further by reproducing phenotypes observed for zantrins Z1 and Z3, and demonstrate that the alkaloid berberine, a reported FtsZ inhibitor, exhibits auto-fluorescence, making it incompatible with systems that employ GFP or YFP tagged FtsZ. These studies describe unique examples of nonnucleotide, competitive FtsZ inhibitors that disrupt FtsZ in vivo, together with a model system that should be useful for in vivo testing of FtsZ inhibitor leads that have been identified through in vitro screens but are unable to penetrate the Gram-negative outer membrane.
Assuntos
Antibacterianos/química , Proteínas de Bactérias/antagonistas & inibidores , Compostos de Benzil/química , Proteínas do Citoesqueleto/antagonistas & inibidores , Éteres Cíclicos/química , Amidoidrolases/genética , Amidoidrolases/metabolismo , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Compostos de Benzil/síntese química , Compostos de Benzil/isolamento & purificação , Compostos de Benzil/farmacologia , Berberina/química , Berberina/farmacologia , Proteínas do Citoesqueleto/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Éteres Cíclicos/isolamento & purificação , Éteres Cíclicos/farmacologia , GTP Fosfo-Hidrolases/metabolismo , Bactérias Gram-Positivas/efeitos dos fármacos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genéticaRESUMO
Self-assembling protein filaments are at the heart of cell function. Among them, tubulin-like proteins are essential for cell division, DNA segregation and cytoskeletal functions across the domains of life. FtsZ and tubulin share their core structures, a characteristic nucleotide-binding pocket and similar protofilament architecture. GTP hydrolysis between consecutive subunits drives their assembly dynamics. Two recent studies provide previously missing, filament atomic structures of bacterial FtsZ and a recently discovered archaeal tubulin in their nucleotide triphosphate-bound states. Both filament structures reveal strikingly conserved interfacial GTPase active sites, with Mg2+ and K+ /Na+ cations and an NxDxxD/E triad of catalytic residues, probably inherited from the common ancestor of FtsZs and tubulins. Moreover, both proteins exhibit nucleotide-regulated subunit association mediated by interfacial water bridges, as well as polymerization-induced structural changes, likely enabling related dynamic assembly mechanisms.
Assuntos
GTP Fosfo-Hidrolases , Tubulina (Proteína) , Tubulina (Proteína)/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Proteínas do Citoesqueleto/química , Archaea/genética , Archaea/metabolismo , Proteínas de Bactérias/metabolismo , Bactérias/metabolismo , Nucleotídeos , Guanosina Trifosfato/metabolismoRESUMO
The structure of the unique bacterial tubulin BtubA/B from Prosthecobacter is very similar to eukaryotic αß-tubulin but, strikingly, BtubA/B fold without eukaryotic chaperones. Our sequence comparisons indicate that BtubA and BtubB do not really correspond to either α- or ß-tubulin but have mosaic sequences with intertwining features from both. Their nucleotide-binding loops are more conserved, and their more divergent sequences correspond to discrete surface zones of tubulin involved in microtubule assembly and binding to eukaryotic cytosolic chaperonin, which is absent from the Prosthecobacter dejongeii draft genome. BtubA/B cooperatively assembles over a wider range of conditions than αß-tubulin, forming pairs of protofilaments that coalesce into bundles instead of microtubules, and it lacks the ability to differentially interact with divalent cations and bind typical tubulin drugs. Assembled BtubA/B contain close to one bound GTP and GDP. Both BtubA and BtubB subunits hydrolyze GTP, leading to disassembly. The mutant BtubA/B-S144G in the tubulin signature motif GGG(T/S)G(S/T)G has strongly inhibited GTPase, but BtubA-T147G/B does not, suggesting that BtubB is a more active GTPase, like ß-tubulin. BtubA/B chimera bearing the ß-tubulin loops M, H1-S2, and S9-S10 in BtubB fold, assemble, and have reduced GTPase activity. However, introduction of the α-tubulin loop S9-S10 with its unique eight-residue insertion impaired folding. From the sequence analyses, its primitive assembly features, and the properties of the chimeras, we propose that BtubA/B were acquired shortly after duplication of a spontaneously folding α- and ß-tubulin ancestor, possibly by horizontal gene transfer from a primitive eukaryotic cell, followed by divergent evolution.
Assuntos
Proteínas de Bactérias/genética , Células Eucarióticas/fisiologia , Evolução Molecular , Transferência Genética Horizontal/fisiologia , Bactérias Gram-Negativas/fisiologia , Dobramento de Proteína , Tubulina (Proteína)/genética , Substituição de Aminoácidos , Proteínas de Bactérias/metabolismo , Guanosina Difosfato/genética , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/genética , Guanosina Trifosfato/metabolismo , Mutação de Sentido Incorreto , Estrutura Secundária de Proteína , Tubulina (Proteína)/metabolismoRESUMO
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.
RESUMO
The molecular switch for nucleotide-regulated assembly and disassembly of the main prokaryotic cell division protein FtsZ is unknown despite the numerous crystal structures that are available. We have characterized the functional motions in FtsZ with a computational consensus of essential dynamics, structural comparisons, sequence conservation, and networks of co-evolving residues. Employing this information, we have constructed 17 mutants, which alter the FtsZ functional cycle at different stages, to modify FtsZ flexibility. The mutant phenotypes ranged from benign to total inactivation and included increased GTPase, reduced assembly, and stabilized assembly. Six mutations clustering at the long cleft between the C-terminal beta-sheet and core helix H7 deviated FtsZ assembly into curved filaments with inhibited GTPase, which still polymerize cooperatively. These mutations may perturb the predicted closure of the C-terminal domain onto H7 required for switching between curved and straight association modes and for GTPase activation. By mapping the FtsZ assembly switch, this work also gives insight into FtsZ druggability because the curved mutations delineate the putative binding site of the promising antibacterial FtsZ inhibitor PC190723.
Assuntos
Proteínas de Bactérias/química , Divisão Celular , Proteínas do Citoesqueleto/química , Análise Mutacional de DNA/métodos , Modelos Moleculares , Mutação/genética , Sequência de Aminoácidos , Proteínas de Bactérias/ultraestrutura , Proteínas do Citoesqueleto/ultraestrutura , Evolução Molecular , Luz , Proteínas Mutantes/química , Maleabilidade , Estrutura Secundária de Proteína , Espalhamento de Radiação , Análise de Sequência de ProteínaRESUMO
Tubulin is able to switch between a straight microtubule-like structure and a curved structure in complex with the stathmin-like domain of the RB3 protein (T(2)RB3). GTP hydrolysis following microtubule assembly induces protofilament curvature and disassembly. The conformation of the labile tubulin heterodimers is unknown. One important question is whether free GDP-tubulin dimers are straightened by GTP binding or if GTP-tubulin is also curved and switches into a straight conformation upon assembly. We have obtained insight into the bending flexibility of tubulin by analyzing the interplay of tubulin-stathmin association with the binding of several small molecule inhibitors to the colchicine domain at the tubulin intradimer interface, combining structural and biochemical approaches. The crystal structures of T(2)RB3 complexes with the chiral R and S isomers of ethyl-5-amino-2-methyl-1,2-dihydro-3-phenylpyrido[3,4-b]pyrazin-7-yl-carbamate, show that their binding site overlaps with colchicine ring A and that both complexes have the same curvature as unliganded T(2)RB3. The binding of these ligands is incompatible with a straight tubulin structure in microtubules. Analytical ultracentrifugation and binding measurements show that tubulin-stathmin associations (T(2)RB3, T(2)Stath) and binding of ligands (R, S, TN-16, or the colchicine analogue MTC) are thermodynamically independent from one another, irrespective of tubulin being bound to GTP or GDP. The fact that the interfacial ligands bind equally well to tubulin dimers or stathmin complexes supports a bent conformation of the free tubulin dimers. It is tempting to speculate that stathmin evolved to recognize curved structures in unassembled and disassembling tubulin, thus regulating microtubule assembly.
Assuntos
Microtúbulos , Multimerização Proteica , Estatmina/química , Tubulina (Proteína)/química , Animais , Cristalografia por Raios X , Humanos , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Ovinos , Estatmina/agonistas , Estatmina/metabolismo , Tubulina (Proteína)/agonistas , Tubulina (Proteína)/metabolismoRESUMO
Cell division protein FtsZ can form single-stranded filaments with a cooperative behavior by self-switching assembly. Subsequent condensation and bending of FtsZ filaments are important for the formation and constriction of the cytokinetic ring. PC190723 is an effective bactericidal cell division inhibitor that targets FtsZ in the pathogen Staphylococcus aureus and Bacillus subtilis and does not affect Escherichia coli cells, which apparently binds to a zone equivalent to the binding site of the antitumor drug taxol in tubulin (Haydon, D. J., Stokes, N. R., Ure, R., Galbraith, G., Bennett, J. M., Brown, D. R., Baker, P. J., Barynin, V. V., Rice, D. W., Sedelnikova, S. E., Heal, J. R., Sheridan, J. M., Aiwale, S. T., Chauhan, P. K., Srivastava, A., Taneja, A., Collins, I., Errington, J., and Czaplewski, L. G. (2008) Science 312, 1673-1675). We have found that the benzamide derivative PC190723 is an FtsZ polymer-stabilizing agent. PC190723 induced nucleated assembly of Bs-FtsZ into single-stranded coiled protofilaments and polymorphic condensates, including bundles, coils, and toroids, whose formation could be modulated with different solution conditions. Under conditions for reversible assembly of Bs-FtsZ, PC190723 binding reduced the GTPase activity and induced the formation of straight bundles and ribbons, which was also observed with Sa-FtsZ but not with nonsusceptible Ec-FtsZ. The fragment 2,6-difluoro-3-methoxybenzamide also induced Bs-FtsZ bundling. We propose that polymer stabilization by PC190723 suppresses in vivo FtsZ polymer dynamics and bacterial division. The biochemical action of PC190723 on FtsZ parallels that of the microtubule-stabilizing agent taxol on the eukaryotic structural homologue tubulin. Both taxol and PC190723 stabilize polymers against disassembly by preferential binding to each assembled protein. It is yet to be investigated whether both ligands target structurally related assembly switches.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/metabolismo , Excipientes/farmacologia , Piridinas/farmacologia , Tiazóis/farmacologia , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/enzimologia , Bacillus subtilis/crescimento & desenvolvimento , Sítios de Ligação , Divisão Celular/efeitos dos fármacosRESUMO
We describe here an extraordinary purple-colored DNA ligase, LigFa, from the acidophilic ferrous iron-oxidizing archaeon Ferroplasma acidiphilum, a di-ferric enzyme with an extremely low pH activity optimum. Unlike any other DNA ligase studied to date, LigFa contains two Fe(3+)-tyrosinate centers and lacks any requirement for either Mg(2+) or K(+) for activity. DNA ligases from closest phylogenetic and ecophysiological relatives have normal pH optima (6.0-7.5), lack iron, and require Mg(2+)/K(+) for activity. Ferric iron retention is pH-dependent, with release resulting in partial protein unfolding and loss of activity. Reduction of the Fe(3+) to Fe(2+) results in an 80% decrease in DNA substrate binding and an increase in the pH activity optimum to 5.0. DNA binding induces significant conformational change around the iron site(s), suggesting that the ferric irons of LigFa act both as structure organizing and stabilizing elements and as Lewis acids facilitating DNA binding at low pH.
Assuntos
DNA Ligases/metabolismo , Ferro/metabolismo , Thermoplasmales/enzimologia , Sequência de Aminoácidos , DNA/metabolismo , DNA Ligase Dependente de ATP , DNA Ligases/química , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Mutação , Conformação Proteica , Proteínas Recombinantes/metabolismo , Espectroscopia de Mossbauer , Especificidade por SubstratoRESUMO
Bacterial resistance to antibiotics makes previously manageable infections again disabling and lethal, highlighting the need for new antibacterial strategies. In this regard, inhibition of the bacterial division process by targeting key protein FtsZ has been recognized as an attractive approach for discovering new antibiotics. Binding of small molecules to the cleft between the N-terminal guanosine triphosphate (GTP)-binding and the C-terminal subdomains allosterically impairs the FtsZ function, eventually inhibiting bacterial division. Nonetheless, the lack of appropriate chemical tools to develop a binding screen against this site has hampered the discovery of FtsZ antibacterial inhibitors. Herein, we describe the first competitive binding assay to identify FtsZ allosteric ligands interacting with the interdomain cleft, based on the use of specific high-affinity fluorescent probes. This novel assay, together with phenotypic profiling and X-ray crystallographic insights, enables the identification and characterization of FtsZ inhibitors of bacterial division aiming at the discovery of more effective antibacterials.
Assuntos
Antibacterianos/química , Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/metabolismo , Sítio Alostérico , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Benzamidas/química , Benzamidas/metabolismo , Benzamidas/farmacologia , Cristalografia por Raios X , Proteínas do Citoesqueleto/antagonistas & inibidores , Polarização de Fluorescência , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Ligantes , Testes de Sensibilidade Microbiana , Ligação Proteica , Piridinas/química , Piridinas/metabolismo , Piridinas/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo , Relação Estrutura-AtividadeRESUMO
Essential cell division protein FtsZ forms the bacterial cytokinetic ring and is a target for new antibiotics. FtsZ monomers bind GTP and assemble into filaments. Hydrolysis to GDP at the association interface between monomers leads to filament disassembly. We have developed a homogeneous competition assay, employing the fluorescence anisotropy change of mant-GTP upon binding to nucleotide-free FtsZ, which detects compounds binding to the nucleotide site in FtsZ monomers and measures their affinities within the millimolar to 10 nM range. We have employed this method to determine the apparent contributions of the guanine, ribose, and the α-, ß-, and γ-phosphates to the free energy change of nucleotide binding. Similar relative contributions have also been estimated through molecular dynamics and binding free energy calculations, employing the crystal structures of FtsZ-nucleotide complexes. We find an energetically dominant contribution of the ß-phosphate, comparable to the whole guanosine moiety. GTP and GDP bind with similar observed affinity to FtsZ monomers. Loss of the regulatory γ-phosphate results in a predicted accommodation of GDP which has not been observed in the crystal structures. The binding affinities of a series of C8-substituted GTP analogues, known to inhibit FtsZ but not eukaryotic tubulin assembly, correlate with their inhibitory capacity on FtsZ polymerization. Our methods permit testing of FtsZ inhibitors targeting its nucleotide site, as well as compounds from virtual screening of large synthetic libraries. Our results give insight into the FtsZ-nucleotide interactions, which could be useful in the rational design of new inhibitors, especially GTP phosphate mimetics.