Interacciones de la proteína de división celular FtsZ con nucleótidos e inhibidores : en busca de nuevos antibióticos

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Biológicas, Departamento de Bioquímica y Biología Molecular I, leída el 02-12-2010Depto. de Bioquímica y Biología MolecularFac. de Ciencias BiológicasTRUEunpu

Interacciones de la proteína de división celular FtsZ con nucleótidos e inhibidores. En busca de nuevos antibióticos.

149 p.-40 fig.-10 tab.7 A tab.FtsZ es la proteína fundamental de la división celular bacteriana. FtsZ autoensambla formando protofilamentos que se asocian en el anillo citoesquelético. Forma parte de la superfamilia de GTPasas citoesqueléticas de la tubulina, que también incluye a αβ-tubulina, a γ-tubulina, tubulina bacteriana y TubZ.FtsZ es una diana para la búsqueda de pequeñas moléculas inhibidoras que podrían constituir una nueva clase de compuestos antibacterianos con los que combatir la emergencia continua de bacterias patógenas resistentes a los antibióticos actuales. El conocimiento de las interacciones de FtsZ con nucleótidos de guanina aporta información sobre los mecanismos funcionales de esta proteína, que puede ser muy útil para el diseño de inhibidores específicos de FtsZ. Además, un ensayo que permita medir la unión de cualesquiera moléculas que reemplacen a GTP de FtsZ podría ser utilizado para buscar nuevos inhibidores de esta proteína.En esta Tesis se ha desarrollado en primer lugar un método de competición, empleando el nucleótido fluorescente mant-GTP junto con apoFtsZ, que permite la detección y la medida de la afinidad de moléculas que se unen al sitio de nucleótido de FtsZ. Se ha comparado con un método de competición complementario, basado en el desplazamiento de 3H-GTP, para validar las afinidades medidas.A continuación, se ha utilizado el método fluorescente de competición para estudiar la unión de los ligandos reguladores GTP y GDP a su sitio en FtsZ. Para ello se midieron las uniones de los componentes del GTP y de otros nucleótidos. Se han realizado simulaciones de dinámica molecular para analizar el tipo y localización de las interacciones entre los nucleótidos y FtsZ. Se ha encontrado que la interacción con el fosfato-β tiene la mayor relevancia energética, contribuyendo por sí sola en casi igual medida que la guanosina. Además, se propone que la presencia del fosfato-γ provoca un reordenamiento de la proteína y el nucleótido, estableciéndose interacciones diferentes que no conllevan un cambio significativo de la afinidad observada del GTP respecto a la del GDP.Finalmente, se investigaron los efectos de análogos del GTP y de compuestos químicamente diferentes. He observado que la capacidad de inhibición de la polimerización por análogos del GTP sustituidos en C8 se correlaciona con su afinidad por la proteína. También se ensayaron una serie de compuestos elegidos por cribado virtual, además de un péptido con supuestos efectos inhibidores sobre FtsZ, con resultados negativos en ambos casos. Por último se estudió el mecanismo de acción de PC190723, un compuesto que protege frente a una dosis letal de Staphylococus aureus en un modelo murino y que tiene como diana a FtsZ. PC190723 es un estabilizador de polímeros de FtsZ, lo que probablemente explica su efecto inhibidor de la división celular en especies bacterianas sensibles.Peer reviewe

Targetting assembly of cell division protein FtsZ with small molecules

9 páginas, 4 figuras, 1 tabla -- PAGS nros. 269-277FtsZ is the key protein of bacterial cell division and an emergent target for new antibiotics. It is a filament-forming GTPase and a structural homologue of eukaryotic tubulin. A number of FtsZ-interacting compounds have been reported, some of which have powerful antibacterial activity. Here we review recent advances and new approaches in modulating FtsZ assembly with small molecules. This includes analyzing their chemical features, binding sites, mechanisms of action, the methods employed, and computational insights, aimed at a better understanding of their molecular recognition by FtsZ and at rational antibiotic design. Cell division protein FtsZ is employed by most bacteria to divide. It forms the cytokinetic Z-ring at the division site(1) (Figure 1), is tethered to the inner face of the plasma membrane by FtsA and ZipA, and recruits other accessory proteins of the cell division machinery (divisome), several of which are essential for remodeling the cell wall peptidoglycan at the septum.(2, 3) The assembly of the Z-ring is coordinated with DNA segregation and cell growth through many regulatory proteins in different bacteria.(2) The bacterial division proteins differ from the proteins of eukaryotic cytokinesis. Thus, bacterial cytoskeleton and cell division have been recognized as attractive targets for seeking new antibiotics(4, 5) with which to fight the widespread emergence of pathogens resistant to current antibiotics.(6) FtsZ has been validated as a target for antibacterial intervention with a synthetic compound active on an in vivo model of infection.(7) Very recently, it has been found that antibiotic acyldepsipeptides activate bacterial ClpP peptidase to degrade FtsZ.(8) Here we review small molecule approaches for targeting FtsZ assemblyWork in the authors’ laboratories has been supported by grants MICINN BFU2008-00013, BFU2009-09552 and SAF2010-22198 and grants CAM S-BIO-0214-2006 and S-SAL-249-2006Peer reviewe

Targeting the Assembly of Bacterial Cell Division Protein FtsZ with Small Molecules

FtsZ is the key protein of bacterial cell division and an emergent target for new antibiotics. It is a filament-forming GTPase and a structural homologue of eukaryotic tubulin. A number of FtsZ-interacting compounds have been reported, some of which have powerful antibacterial activity. Here we review recent advances and new approaches in modulating FtsZ assembly with small molecules. This includes analyzing their chemical features, binding sites, mechanisms of action, the methods employed, and computational insights, aimed at a better understanding of their molecular recognition by FtsZ and at rational antibiotic design

The interactions of cell division protein FtsZ with guanine nucleotides

14 páginas, 10 figuras, 5 tablas -- PAGS nros. 37515-37528Prokaryotic 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 Kb values ranging from 20 to 300 μm-1 under various conditions. GTP·Mg2+ and GDP·Mg2+ bind with slightly reduced affinity. Bound GTP and the coordinated Mg2+ ion play a minor structural role in FtsZ monomers, but Mg2+-assisted GTP hydrolysis triggers polymer disassembly. Mant-GTP binds and dissociates quickly from FtsZ monomers, with ∼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 ∼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. Previous SectionNext SectionFtsZ is a cytoskeletal protein essential to bacterial cytokinesis and a member of the tubulin family of GTPases, which also includes αβ-tubulin (1), γ-tubulin (2), bacterial tubulin BtubA/B (3, 4), and TubZ (5). FtsZ assembles by forming filaments that constitute the Z-ring at the cell division site in bacteria. The Z-ring, a dynamic structure maintained by assembly and disassembly of FtsZ, recruits the other elements of the division machinery following chromosome segregation (6-10). Bacterial cell growth and division are regulated by nutrient availability; a metabolic sensor has been recently identified in Bacillus subtilis, including an effector, the glucosyltransferase UgtP, which modulates FtsZ assembly (11). GTP binding, hydrolysis, and exchange constitute the regulatory mechanism responsible for dynamics of FtsZ and tubulin polymers. The nucleotide switches of these assembling GTPases appear to involve polymerization-driven structural changes (12), although FtsZ and tubulin form different end polymers. The GTPase activity of FtsZ is modified by the polymerization inhibitory protein MipZ (13) and, weakly, by EzrA (14).The hydrolyzable nucleotide bound to tubulin becomes occluded in microtubule protofilaments (15). Microtubules hydrolyze all bound GTP to GDP except at their very ends and become metastable, giving rise to microtubule dynamic instability (16). In contrast, polymers of FtsZ from E. coli were reported to contain mostly GTP, and, under certain conditions, nucleotide exchange proceeds faster than hydrolysis (17). This suggested that the nucleotide binding site remains exchangeable in FtsZ polymers, which would therefore be devoid of dynamic instability. Polymers of Methanococcus jannaschii FtsZ were found to contain different proportions of GTP and GDP (depending on the hydrolysis rate) and to rapidly depolymerize upon either GTP consumption or GDP addition (18, 19). GDP binding destabilizes M. jannaschhi FtsZ polymers compared with polymers with GTP or without a bound nucleotide (20). In E. coli FtsZ polymers the main rate-limiting step in nucleotide turnover was found to be nucleotide hydrolysis, rapidly followed by phosphate release, whereas a second rate-limiting step could be nucleotide dissociation. However, whether nucleotide dissociation took place directly from the polymer or through depolymerization into subunits, followed by GDP release, was not determined (21). An important problem yet to be solved for FtsZ assembly dynamics is whether, following GTP hydrolysis (i) GDP dissociates from subunits in the FtsZ polymer which directly reload with GTP, (ii) polymer subunits exchange with GTP-bound subunits in solution, or (iii) the FtsZ-GDP polymer fully disassembles and reassembles again from GTP-bound subunits. Consistent with an exchangeable nucleotide in FtsZ polymers, the nucleotide was observed to be largely accessible in the crystal structure of a protofilament-like dimer of M. jannaschii FtsZ (22). On the other hand, exchange of GFP-FtsZ fusions in bacterial Z-rings was found to proceed with a half-time of 8-9 s in vivo, by means of fluorescence recovery after photobleaching (23, 24). As observed in an in vitro fluorescence resonance energy transfer assay, subunit turnover in filaments of E. coli FtsZ took place with a half-time of 7 s with GTP, which was slowed down under conditions reducing the nucleotide hydrolysis rate (25). This rate of subunit turnover is comparable with the turnover rate of GTP hydrolysis (21) and with the rate of depolymerization in GDP excess, suggesting that GDP does not exchange into intact filaments (23). This favors the interpretation that the rapid assembly dynamics of FtsZ filaments may operate by a mechanism related to microtubule dynamic instability (25). In addition, subunit turnover and GTPase in FtsZ from Mycobacterium tuberculosis are both about 10 times slower than in E. coli FtsZ (26).FtsZ and its nucleotide binding site are attractive targets for cell division inhibitors, which may lead to new classes of antibacterial compounds (27) to fight the continuous emergence of antibiotic resistance. Small molecules reported to modulate FtsZ assembly include 8-bromo-GTP (28) and other nucleotide analogues (29), 3-metoxybenzamide (30), viriditoxin (31), ruthenium red (32), zantrins (33), SRI-3072 (34), polyphenols (35), PC58538 and PC170942 (36), sanguinarine (37), certain taxanes (38), A189 (39), amikacin (40), totarol (41), and cinnamalehyde (42). This study focused on fundamental processes of FtsZ-nucleotide interactions. We have determined the energetics of GTP and GDP binding to FtsZ and the kinetics of binding and dissociation in FtsZ monomers and polymers using fluorescent (mant)6-nucleotides. The results reveal functional differences with nucleotide binding to tubulin that will facilitate screening for compounds binding to the nucleotide site of FtsZ. They also indicate a slowed down nucleotide exchange in FtsZ polymers, which provides insight to their dynamicsThis work was supported in part by grants Ministerio de Educacion y Ciencia (MEC) BFU 2005-00505/BMC (to J. M. A.), MEC BFU 2006-10288 (to M. M.), and Comunidad Autónoma de Madrid S-BIO-0214-2006 (to J. M. A. and J. F. D.), a Consejo Superior de Investigaciones Científicas-I3P postdoctoral contract (to S. H.), a MEC-FPI predoctoral fellowship (to C. S.), and Travel Grant FAPESP CBME-98/14138-2 (to W. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.Peer reviewe

Insights into nucleotide recognition by cell division protein FtsZ from a mant-GTP competition assay and molecular dynamics.

Supporting information of this article: Table of FtsZ inhibitors (S1), tables of MM-GBSA energies (S2-S7), tables of virtual screening compounds (S8 and S9), Figures S1-S3, and PDB files with average structures from molecular dynamics, can be found free of charge via the Internet at http://pubs.acs.org/doi/full/10.1021/bi101577pEssential 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-, β-, 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 β-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.This work was supported by grants from the Spanish Ministry for Science (MCINN) BFU2008-00013 (J.M.A.), Madrid Community S-BIO-0214-2006 (A.M. and J.M.A.), and “Vernieuwingsimpuls” Grant 016.001.024, (T.d.B.) of The Netherlands Organization for Scientific Research (NWO). C.S.-B. and L.B.R.-A. had FPI predoctoral fellowships. R.G.-R. had a contract from MCINN “Programa de Personal Técnico y de Apoyo 2008”. We thank OpenEye Scientific Software, Inc., for providing us with academic license for their software. Barcelona Supercomputing Center is acknowledged for the generous allocation of computer time.Peer reviewe

The Antibacterial Cell Division Inhibitor PC190723 Is an FtsZ Polymer-stabilizing Agent That Induces Filament Assembly and Condensation*

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

Probing FtsZ and tubulin with C-8-substituted GTP analogs reveals differences in their nucleotide binding sites

11 páginas, 4 figuras, 2 tablas -- PAGS nros. 189-199The 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 chainThis work was supported in part by a Netherlands Organization for Scientific Research “Vernieuwingsimpuls” grant (016.001.024, T.d.B.) and grants MEC BFU 2005-00505/BMC and CAM S-BIO-0214-2006 (J.M.A.). A.V.P. acknowledges support by the Avenir grant of Inserm, ACI BCMS of the French Research Ministry (project BCM0210), and equipment grants of the “La Ligue contre le Cancer” (Comité de l'Isère) and the “Association pour la Recherche sur le Cancer” (project 7833). A.J. was funded by the program “Emergence 2004” of the Department de Rhône-AlpesPeer reviewe