Dinámica de los filamentos de Ftsz y búsqueda racional de inhibidores sintéticos con actividad antibacteriana

Resum

The cytoskeletal protein FtsZ forms filaments that organize into the bacterial Z-ring, which recruits other cell-division proteins and helps provide some of the constrictive force for cytokinesis (Osawa et al., 2008). Given its ubiquity and its central role in bacterial cell division, FtsZ is an attractive target for the development of new antibiotics (Schaffner-Barbero et al., 2012). FtsZ is composed of two globular subdomains, connected by a long helix (H7) sandwiched between them. Furthermore, FtsZ contains an unstructured extreme C-terminal implicated in interactions between FtsZ and modulatory proteins (Buske and Levin, 2012). Similar to its eukaryotic relative tubulin, FtsZ form protofilaments with GTP (Nogales et al., 1998) and has a built-in molecular switch that is triggered by nucleotide γ-phosphate hydrolysis and permits assembly-disassembly regulation. The GTPase catalytic site is formed during filament assembly (Lowe and Amos, 1999). Monomers of FtsZ can exist in two states: the inactive state with low affinity to form polymers and the active state with high affinity for polymerization (Chen and Erickson, 2011, Huecas et al., 2008, Martin-Galiano et al., 2010). The inactive form of FtsZ monomers has been observed in most crystal structures from different species (Oliva et al., 2007), with the cleft between N and C-terminal domains in a closed conformation. The crystal packing from the recently reported Staphylococcus aureus FtsZ (SaFtsZ) structure, (Elsen et al., 2012, Tan et al., 2012, Matsui et al., 2012) exhibits a straight filament formed by open-cleft monomers. A structural comparison of SaFtsZ with other FtsZs supports the cleft opening mechanism of the FtsZ assembly switch. However, proof involving the same FtsZ protein, such as a closed-cleft monomeric structure of SaFtsZ molecule or an open filamentous structure of other FtsZs, is required. The functional importance of FtsZ makes it an interesting drug target for treating infections caused by bacteria. The main cavities available for ligands binding in an FtsZ monomer are the nucleotide-binding cup in the N-terminal domain and the cleft between N and C-terminal domains. The antibiotic PC190723 binds into the cleft and modulates FtsZ assembly by stabilizing its polymers (Andreu et al., 2010, Elsen et al., 2012). FtsZ has been validated as the target of the in vivo effective antibacterial compound PC190723 (Haydon et al., 2008). In this thesis, we study the FtsZ 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 neighboring monomer in the 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. These molecular dynamics 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. Also, we have collaborated on the design of fluorescent analogs of PC190723 to probe the FtsZ structural assembly switch. Among them, nitrobenzoxadiazole (NBD) probes specifically bind to assembled FtsZ rather than to monomers. 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. These results demonstrate the structural mechanism of the FtsZ assembly switch. Molecular dynamic simulations of SaFtsZ model complexes with the NBD probes indicated the binding mode of these compounds and suggest a binding site extension along the interdomain cleft. Moreover, we have employed these fluorescent probes to set up a fluorescence anisotropy based displacement assay able to detect new allosteric modulators of FtsZ. The structural features of FtsZ polymers of B. subtilis (BsFtsZ) and truncated BsFtsZ (BsFtsZ-∆t), lacking the entire C-terminal tail, have been studied by SAXS and Cryo-electron microscopy. BsFtsZ-∆t showed several striking differences respecting to the full-length protein polymers. Experiments indicate that BsFtsZ polymers are formed by aggregation or annealing of curved filaments bundles with lateral spacing (~70 Å) between neighboring protofilaments. However, the truncated protein filaments are held straight in bundles by their close lateral contacts. We propose that the 70 Å spacing between BsFtsZ filaments forming loose bundles is provided by the C-terminal extension. The parallel BsFtsZ-∆t filaments possibly stick to each other generating the straight bundles, whereas a flexible lateral association allows filament curvature in the full length BsFtsZ. Virtual screening of large compound libraries is a popular approach to identify new antibacterial agents. Using this technique, it has been identified several hits compounds for the nucleotide-binding site (UCM05 y VS18), however, the compounds tested in the binding site PC, with different structures did not show effect on cells. UCM05 interacts with the nucleotide-binding site of BsFtsZ (Kd = 2.3 μM) and inhibits the growth of the Gram-positive bacterium B. subtilis (MIC = 100 μM). In order to improve the binding affinity and the antibacterial efficacy of UCM05, new derivatives selected by docking and molecular dynamics experiments were synthetized. The change in the naphthalene core for biphenyl improves the binding affinity and antibacterial efficacy compared to UCM05. For instance, biphenyl derivative 28 stands out as a potent FtsZ inhibitor (Kd = 0.5 μM) with high antibacterial activity [MIC (B. subtilis) = 5 μM and MIC (MRSA) = 7 μM]. VS18 interacts with the nucleotide-binding site of BsFtsZ (Kd ≈ 90.9 μM). However, VS18 does not have antibacterial activity on B. subtilis (MIC > 600 μM). Its chemical structure is used as a starting point for chemical modifications in virtual screening. Of the selected compounds, 29 improve affinity for the binding site respecting to VS18, validating the protocol followed. The most promising compound is el derivado VS33, which replaces the nucleotide (Kd ≈ 6.7 μM) and affects bacterial-cell division [MIC (B. subtilis) = 50 μM and MIC (MRSA) = 20 μM]. The possible binding modes of compounds 28 and VS33 were analyzed by docking and molecular dynamics simulations studies. They provided a simple explanation of the competitive inhibition mechanism for these compounds. Bibliography ANDREU, J. M., SCHAFFNER-BARBERO, C., HUECAS, S., ALONSO, D., LOPEZ-RODRIGUEZ, M. L., RUIZ-AVILA, L. B., NUNEZ-RAMIREZ, R., LLORCA, O. & MARTIN-GALIANO, A. J. 2010. The antibacterial cell division inhibitor PC190723 is an FtsZ polymer-stabilizing agent that induces filament assembly and condensation. J Biol Chem, 285, 14239-46. BUSKE, P. J. & LEVIN, P. A. 2012. Extreme C terminus of bacterial cytoskeletal protein FtsZ plays fundamental role in assembly independent of modulatory proteins. J Biol Chem, 287, 10945-57. CHEN, Y. & ERICKSON, H. P. 2011. Conformational changes of FtsZ reported by tryptophan mutants. Biochemistry, 50, 4675-84. ELSEN, N. L., LU, J., PARTHASARATHY, G., REID, J. C., SHARMA, S., SOISSON, S. M. & LUMB, K. J. 2012. Mechanism of action of the cell-division inhibitor PC190723: modulation of FtsZ assembly cooperativity. J Am Chem Soc, 134, 12342-5. 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. & CZAPLEWSKI, L. G. 2008. An inhibitor of FtsZ with potent and selective anti-staphylococcal activity. Science, 321, 1673-5. HUECAS, S., LLORCA, O., BOSKOVIC, J., MARTIN-BENITO, J., VALPUESTA, J. M. & ANDREU, J. M. 2008. Energetics and geometry of FtsZ polymers: nucleated self-assembly of single protofilaments. Biophys J, 94, 1796-806. LOWE, J. & AMOS, L. A. 1999. Tubulin-like protofilaments in Ca2+-induced FtsZ sheets. EMBO J, 18, 2364-71. MARTIN-GALIANO, A. J., BUEY, R. M., CABEZAS, M. & ANDREU, J. M. 2010. Mapping flexibility and the assembly switch of cell division protein FtsZ by computational and mutational approaches. J Biol Chem, 285, 22554-65. MATSUI, T., YAMANE, J., MOGI, N., YAMAGUCHI, H., TAKEMOTO, H., YAO, M. & TANAKA, I. 2012. Structural reorganization of the bacterial cell-division protein FtsZ from Staphylococcus aureus. Acta Crystallogr D Biol Crystallogr, 68, 1175-88. NOGALES, E., DOWNING, K. H., AMOS, L. A. & LOWE, J. 1998. Tubulin and FtsZ form a distinct family of GTPases. Nat Struct Biol, 5, 451-8. OLIVA, M. A., TRAMBAIOLO, D. & LOWE, J. 2007. Structural insights into the conformational variability of FtsZ. J Mol Biol, 373, 1229-42. OSAWA, M., ANDERSON, D. E. & ERICKSON, H. P. 2008. Reconstitution of contractile FtsZ rings in liposomes. Science, 320, 792-4. SCHAFFNER-BARBERO, C., MARTIN-FONTECHA, M., CHACON, P. & ANDREU, J. M. 2012. Targeting the assembly of bacterial cell division protein FtsZ with small molecules. ACS Chem Biol, 7, 269-77. TAN, C. M., THERIEN, A. G., LU, J., LEE, S. H., CARON, A., GILL, C. J., LEBEAU-JACOB, C., BENTON-PERDOMO, L., MONTEIRO, J. M., PEREIRA, P. M., ELSEN, N. L., WU, J., DESCHAMPS, K., PETCU, M., WONG, S., DAIGNEAULT, E., KRAMER, S., LIANG, L., MAXWELL, E., CLAVEAU, D., VAILLANCOURT, J., SKOREY, K., TAM, J., WANG, H., MEREDITH, T. C., SILLAOTS, S., WANG-JARANTOW, L., RAMTOHUL, Y., LANGLOIS, E., LANDRY, F., REID, J. C., PARTHASARATHY, G., SHARMA, S., BARYSHNIKOVA, A., LUMB, K. J., PINHO, M. G., SOISSON, S. M. & ROEMER, T. 2012. Restoring methicillin-resistant Staphylococcus aureus susceptibility to beta-lactam antibiotics. Sci Transl Med, 4, 126ra35.