José Manuel Andreu

Centro de Investigaciones Biológicas, Madrid, Madrid, Spain

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Publications (72)307.95 Total impact

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    ABSTRACT: The binding of epothilones to dimeric tubulin and to microtubules has been studied by means of biochemical and NMR techniques. We have determined the binding constants of epothilone A (EpoA) and B (EpoB) to dimeric tubulin, which are four orders of magnitude lower than those for microtubules, and we have elucidated the conformation and binding epitopes of EpoA and EpoB when bound to tubulin dimers and microtubules in solution. The determined conformation of epothilones when bound to dimeric tubulin is similar to that found by X-ray crystallographic techniques for the binding of EpoA to the Tubulin/RB3/TTL complex; it is markedly different from that reported for EpoA bound to zinc-induced sheets obtained by electron crystallography. Likewise, only the X-ray structure of EpoA bound to the Tubulin/RB3/TTL complex at the luminal site, but not the electron crystallography structure, is compatible with the results obtained by STD on the binding epitope of EpoA bound to dimeric tubulin, thus confirming that the allosteric change (structuring of the M-loop) is the biochemical mechanism of induction of tubulin assembly by epothilones. TR-NOESY signals of EpoA bound to microtubules have been obtained supporting the interaction with a transient binding site with a fast exchange rate (pore site), consistent with the notion that epothilones access the luminal site through the pore site, as has also been observed for taxanes. Finally, the differences in the tubulin binding affinities of a series of epothilone analogs has been quantitatively explained using the newly determined binding pose and the COMBINE methodology.
    ACS Chemical Biology 02/2014; · 5.44 Impact Factor
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    ABSTRACT: PM060184 belongs to a new family of tubulin-binding agents originally isolated from the marine sponge Lithoplocamia lithistoides. This compound is currently produced by total synthesis and is under evaluation in clinical studies in patients with advanced cancer diseases. It was recently published that PM060184 presents the highest known affinities among tubulin-binding agents, and that it targets tubulin dimers at a new binding site. Here, we show that PM060184 has a potent antitumor activity in a panel of different tumor xenograft models. Moreover, PM060184 is able to overcome P-gp mediated resistance in vivo, an effect that could be related to its high binding affinity for tubulin. To gain insight into the mechanism responsible of the observed antitumor activity, we have characterized its molecular and cellular effects. We have observed that PM060184 is an inhibitor of tubulin polymerization that reduces microtubule dynamicity in cells by 59%. Interestingly, PM060184 suppresses microtubule shortening and growing at a similar extent. This action affects cells in interphase and mitosis. In the first case, the compound induces a disorganisation and fragmentation of the microtubule network and the inhibition of cell migration. In the second case, it induces the appearance of multipolar mitosis and lagging chromosomes at the metaphase plate. These effects correlate with prometaphase arrest and induction of caspase-dependent apoptosis or appearance of cells in a multinucleated interphase-like state unrelated to classical apoptosis pathways. Taken together, these results indicate that PM060184 represents a new tubulin binding agent with promising potential as an anticancer agent.
    Biochemical pharmacology 01/2014; · 4.25 Impact Factor
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    ABSTRACT: 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.
    Bioorganic Chemistry. 01/2014;
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    ABSTRACT: 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 homolog 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 non-hydrolysable 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 to explain 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.
    Journal of the American Chemical Society 09/2013; · 10.68 Impact Factor
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    ABSTRACT: 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.
    Bioorganic & medicinal chemistry 07/2013; · 2.82 Impact Factor
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    ABSTRACT: We have investigated the target and mechanism of action of a new family of cytotoxic small molecules of marine origin. PM050489 and its dechlorinated analogue PM060184 inhibit the growth of relevant cancer cell lines at subnanomolar concentrations. We found that they are highly potent microtubule inhibitors that impair mitosis with a distinct molecular mechanism. They bind with nanomolar affinity to unassembled alphabeta-tubulin dimers and PM050489 binding is inhibited by known Vinca domain ligands. NMR TR-NOESY data indicated that a hydroxyl-containing analogue, PM060327, binds in an extended conformation and STD results define its binding epitopes. Distinctly from vinblastine, these ligands only weakly induce tubulin self-association, in a manner more reminiscent of isohomohalichondrin B than of eribulin. PM050489, possibly acting like a hinge at the association interface between tubulin heterodimers, reshapes Mg2+-induced 42 S tubulin double rings into smaller 19 S single rings made of 7±1 alphabeta-tubulin dimers. PM060184-resistant mutants of Aspergillus nidulans map to beta-tubulin Asn100, suggesting a new binding site different from vinblastine at the associating beta-tubulin end. Inhibition of assembly dynamics by a few ligand molecules at the microtubule plus end would explain the antitumor activity of these compounds, of which PM060184 is undergoing clinical trials.
    ACS Chemical Biology 07/2013; · 5.44 Impact Factor
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    ABSTRACT: 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.
    ACS Chemical Biology 07/2013; · 5.44 Impact Factor
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    ABSTRACT: Colchicine site ligands with indole B rings are potent tubulin polymerization inhibitors. Structural modifications at the indole 3-position of 1-methyl-5-indolyl based isocombretastatins (1,1-diarylethenes) and phenstatins endowed them with anchors for further derivatization and resulted in highly potent compounds. The substituted derivatives displayed potent cytotoxicity against several human cancer cell lines due to tubulin inhibition, as shown by cell cycle analysis, confocal microscopy and tubulin polymerization inhibitory activity studies and promoted cell killing mediated by caspase-3 activation. Binding at the colchicine site was confirmed by means of fluorescence measurements of MTC displacement. Molecular modeling suggests that the tropolone-binding region of the colchicine site of tubulin can adapt to hosting small polar substituents. Isocombretastatins accepted substitutions better than phenstatins, and the highest potencies were achieved for the cyano and hydroxyiminomethyl substituents, with TPI values in the submicromolar range and cytotoxicities in the subnanomolar range. A 3,4,5-trimethoxyphenyl ring usually afforded more potent derivatives than a 2,3,4-trimethoxyphenyl ring.
    Journal of Medicinal Chemistry 03/2013; · 5.61 Impact Factor
  • José M Andreu, María A Oliva
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    ABSTRACT: Bacterial tubulin BtubA/B is a close structural homolog of eukaryotic αβ-tubulin, thought to have originated by transfer of ancestral tubulin genes from a primitive eukaryotic cell to a bacterium, followed by divergent evolution. BtubA and BtubB are easily expressed homogeneous polypeptides that fold spontaneously without eukaryotic chaperone requirements, associate into weak BtubA/B heterodimers and assemble forming tubulin-like protofilaments. These protofilaments coalesce into pairs and bundles, or form five-protofilament tubules proposed to share the architecture of microtubules. Bacterial tubulin is an attractive framework for tubulin engineering. Potential applications include humanizing different sections of bacterial tubulin with the aims of creating recombinant binding sites for antitumor drugs, obtaining well-defined substrates for the enzymes responsible for tubulin posttranslational modification, or bacterial microtubule-like polymeric trails for motor proteins. Several divergent sequences from the surface loops of bacterial tubulin have already been replaced by the corresponding eukaryotic sequences, yielding soluble folded chimeras. We describe the purification protocol of untagged bacterial tubulin BtubA/B by means of ion exchange, size exclusion chromatography, and an assembly-disassembly cycle. This is followed by methods and examples to characterize its assembly, employing light scattering, sedimentation, and electron microscopy.
    Methods in cell biology 01/2013; 115:269-81. · 1.44 Impact Factor
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    ABSTRACT: Several pinacol derivatives of podophyllotoxins bearing different side chains and functions at C-7 were synthesized through reductive cross-coupling of podophyllotoxone and several aldehydes and ketones. While possessing a hydroxylated chain at C-7, the compounds retained their respective hydroxyl group with either the 7α (podo) or 7β (epipodo) configuration. Along with pinacols, some C-7 alkylidene and C-7 alkyl derivatives were also prepared. Cytotoxicities against neoplastic cells followed by cell cycle arrest and cellular microtubule disruption were evaluated and mechanistically characterized through tubulin polymerization inhibition and assays of binding to the colchicine site. Compounds of the epipodopinacol (7β-OH) series behaved similarly to podophyllotoxin in all the assays and proved to be the most potent inhibitors. Significantly, 7α-isopropyl-7-deoxypodophyllotoxin (20), without any hydroxyl function, appeared as a promising lead compound for a novel type of tubulin polymerization inhibitors. Experimental results were in overall agreement with modeling and docking studies performed on representative compounds of each series.
    Journal of Medicinal Chemistry 05/2012; 55(15):6724-37. · 5.61 Impact Factor
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    ABSTRACT: 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.
    Proceedings of the National Academy of Sciences 04/2012; 109(20):7711-6. · 9.74 Impact Factor
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    ABSTRACT: Septins form a conserved family of filament forming GTP binding proteins found in a wide range of eukaryotic cells. They share a common structural architecture consisting of an N-terminal domain, a central GTP binding domain and a C-terminal domain, which is often predicted to adopt a coiled-coil conformation, at least in part. The crystal structure of the human SEPT2/SEPT6/SEPT7 heterocomplex has revealed the importance of the GTP binding domain in filament formation, but surprisingly no electron density was observed for the C-terminal domains and their function remains obscure. The dearth of structural information concerning the C-terminal region has motivated the present study in which the putative C-terminal domains of human SEPT2, SEPT6 and SEPT7 were expressed in E. coli and purified to homogeneity. The thermal stability and secondary structure content of the domains were studied by circular dichroism spectroscopy, and homo- and hetero-interactions were investigated by size exclusion chromatography, chemical cross-linking, analytical ultracentrifugation and surface plasmon resonance. Our results show that SEPT6-C and SEPT7-C are able to form both homo- and heterodimers with a high α-helical content in solution. The heterodimer is elongated and considerably more stable than the homodimers, with a K(D) of 15.8 nM. On the other hand, the homodimer SEPT2-C has a much lower affinity, with a K(D) of 4 μM, and a moderate α-helical content. Our findings present the first direct experimental evidence toward better understanding the biophysical properties and coiled-coil pairings of such domains and their potential role in filament assembly and stability.
    Cell biochemistry and biophysics 03/2012; 62(2):317-28. · 3.34 Impact Factor
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    ABSTRACT: Microtubules assembled with paclitaxel and docetaxel differ in their numbers of protofilaments, reflecting modification of the lateral association between αβ-tubulin molecules in the microtubule wall. These modifications of microtubule structure, through a not-yet-characterized mechanism, are most likely related to the changes in tubulin-tubulin interactions responsible for microtubule stabilization by these antitumor compounds. We have used a set of modified taxanes to study the structural mechanism of microtubule stabilization by these ligands. Using small-angle x-ray scattering, we have determined how modifications in the shape and size of the taxane substituents result in changes in the interprotofilament angles and in their number. The observed effects have been explained using NMR-aided docking and molecular dynamic simulations of taxane binding at the microtubule pore and luminal sites. Modeling results indicate that modification of the size of substituents at positions C7 and C10 of the taxane core influence the conformation of three key elements in microtubule lateral interactions (the M-loop, the S3 β-strand, and the H3 helix) that modulate the contacts between adjacent protofilaments. In addition, modifications of the substituents at position C2 slightly rearrange the ligand in the binding site, modifying the interaction of the C7 substituent with the M-loop.
    Biophysical Journal 12/2011; 101(12):2970-80. · 3.67 Impact Factor
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    ABSTRACT: Cyclostreptin is the first microtubule-stabilizing agent whose mechanism of action was discovered to involve formation of a covalent bond with tubulin. Treatment of cells with cyclostreptin irreversibly stabilizes their microtubules because cyclostreptin forms a covalent bond to β-tubulin at either the T220 or the N228 residue, located at the microtubule pore or luminal taxoid binding site, respectively. Because of its unique mechanism of action, cyclostreptin overcomes P-glycoprotein-mediated multidrug resistance in tumor cells. We used a series of reactive cyclostreptin analogues, 6-chloroacetyl-cyclostreptin, 8-chloroacetyl-cyclostreptin, and [(14)C-acetyl]-8-acetyl-cyclostreptin, to characterize the cellular target of the compound and to map the binding site. The three analogues were cytotoxic and stabilized microtubules in both sensitive and multidrug resistant tumor cells. In both types of cells, we identified β-tubulin as the only or the predominantly labeled cellular protein, indicating that covalent binding to microtubules is sufficient to prevent drug efflux mediated by P-glycoprotein. 6-Chloroacetyl-cyclostreptin, 8-chloroacetyl-cyclostreptin, and 8-acetyl-cyclostreptin labeled both microtubules and unassembled tubulin at a single residue of the same tryptic peptide of β-tubulin as was labeled by cyclostreptin (219-LTTPTYGDLNHLVSATMSGVTTCLR-243), but labeling with the analogues occurred at different positions of the peptide. 8-Acetyl-cyclostreptin reacted with either T220 or N228, as did the natural product, while 8-chloroacetyl-cyclostreptin formed a cross-link to C241. Finally, 6-chloroacetyl-cyclostreptin reacted with any of the three residues, thus labeling the pathway for cyclostreptin-like compounds, leading from the pore where these compounds enter the microtubule to the luminal binding pocket.
    Biochemistry 12/2011; 51(1):329-41. · 3.38 Impact Factor
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    ABSTRACT: 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.
    ACS Chemical Biology 11/2011; 7(2):269-77. · 5.44 Impact Factor
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    ABSTRACT: Septins are a conserved group of GTP-binding proteins that form hetero-oligomeric complexes which assemble into filaments. These are essential for septin function, including their role in cytokinesis, cell division, exocytosis and membrane trafficking. Septin 2 (SEPT2) is a member of the septin family and has been associated with neurofibrillary tangles and other pathological features of senile plaques in Alzheimer's disease. An in silico analysis of the amino acid sequence of SEPT2 identified regions with a significant tendency to aggregate and/or form amyloid. These were all observed within the GTP-binding domain. This was consistent with the experimental identification of a structure rich in β-sheet during temperature induced unfolding transitions observed for both the full length protein and the GTP-binding domain alone. This intermediate state is characterized by irreversible aggregation and has the ability to bind Thioflavin-T, suggesting its amyloid nature. Under electron microscopy, fibers extending for several micrometers in length could be visualized. The results shown in this study support the hypothesis that single septins, when present in excess or with unbalanced stoichiometries, may be unstable and assemble into amyloid-like structures.
    Biochimie 09/2011; 94(3):628-36. · 3.14 Impact Factor
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    ABSTRACT: The binding interactions of two antitumor agents that target the paclitaxel site, docetaxel and discodermolide, to unassembled α/β-tubulin heterodimers and microtubules have been studied using biochemical and NMR techniques. The use of discodermolide as a water-soluble paclitaxel biomimetic and extensive NMR experiments allowed the detection of binding of microtubule-stabilizing agents to unassembled tubulin α/β-heterodimers. The bioactive 3D structures of docetaxel and discodermolide bound to α/β-heterodimers were elucidated and compared to those bound to microtubules, where subtle changes in the conformations of docetaxel in its different bound states were evident. Moreover, the combination of experimental TR-NOE and STD NMR data with CORCEMA-ST calculations indicate that docetaxel and discodermolide target an additional binding site at the pore of the microtubules, which is different from the internal binding site at the lumen previously determined by electron crystallography. Binding to this pore site can then be considered as the first ligand-protein recognition event that takes place in advance of the drug internalization process and interaction with the lumen of the microtubules.
    ACS Chemical Biology 05/2011; 6(8):789-99. · 5.44 Impact Factor
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    ABSTRACT: The structure of the unique bacterial tubulin BtubA/B from Prosthecobacter is very similar to eukaryotic αβ-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 β-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 αβ-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 β-tubulin. BtubA/B chimera bearing the β-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 β-tubulin ancestor, possibly by horizontal gene transfer from a primitive eukaryotic cell, followed by divergent evolution.
    Journal of Biological Chemistry 04/2011; 286(22):19789-803. · 4.65 Impact Factor
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    ABSTRACT: 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-, 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.
    Biochemistry 11/2010; 49(49):10458-72. · 3.38 Impact Factor
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    ABSTRACT: 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.
    Journal of Biological Chemistry 10/2010; 285(41):31672-81. · 4.65 Impact Factor

Publication Stats

1k Citations
307.95 Total Impact Points

Institutions

  • 1998–2014
    • Centro de Investigaciones Biológicas
      Madrid, Madrid, Spain
  • 1991–2012
    • Spanish National Research Council
      • Biological Research Centre
      Madrid, Madrid, Spain
  • 2011
    • Spanish National Centre for Cardiovascular Research
      Madrid, Madrid, Spain
  • 2010
    • Aix-Marseille Université
      • Faculté de Pharmacie
      Marseille, Provence-Alpes-Cote d'Azur, France
  • 2006
    • Emory University
      • Winship Cancer Institute
      Atlanta, GA, United States
  • 2003
    • National Center for Biotechnology (CNB)
      Madrid, Madrid, Spain
  • 2002
    • University of Santiago, Chile
      • Facultad de Química y Biología
      Santiago, Region Metropolitana de Santiago, Chile
  • 2000
    • National Institutes of Health
      Maryland, United States