Cytomotive protein assembly machines from the tubulin superfamily of GTPases are essential to divide cells, to segregate DNA or for cytoskeletal and shape functions. Tubulin-like proteins spread in eukaryotes, bacteria, archaea, plasmids and viruses; they include microtubule αβ-tubulin subunits, microtubule organizing γ-tubulin, primitive bacterial tubulin BtubA/B, bacterial cell division protein FtsZ, archaeal shape controlling CetZ and the DNA-positioning TubZs codified by plasmid and phages. They share a few sequence motifs and the same three-dimensional core structure, consisting of an N-terminal GTP binding domain and a GTPase activating domain connected by a central α-helix, but have divergent C-terminal secondary structures and flexible tails. Tubulin-like proteins, except γ-tubulin, typically associate head to tail into polar protofilaments with a 4 nm axial spacing between subunits that form different types of filaments. Their assembly and disassembly is directly linked to their function, forming characteristic subcellular structures such as spindles or division rings. Assembly involves the formation of a subunit-subunit interface where the GTP-binding domain of one subunit in a protofilament interacts with the GTPase activation domain of the next subunit, which complements the GTP pocket and induces GTP hydrolysis. GTP hydrolysis triggers disassembly, which is coupled to free subunits switching back into inactive conformation. Polymer dynamics is based on assembly-disassembly events, shows different features depending on the precise mechanism involved, such as dynamic instability or treadmilling, which can produce motility without the assistance of motor proteins. Tubulin is the target of antitumor drugs that impair microtubule dynamics; FtsZ assembly guides bacterial cell division, which is a target for the discovery of new antibiotics needed to fight resistant pathogens.
Our work focuses on understanding how these protein assembly machines work, how they evolved, and targeting them with small molecules, employing biochemical, crystallography, NMR, computational, microbiological, fluorescence, electron microscopy and synthetic approaches at the CIB and collaborating labs. We have recently demonstated the FtsZ assembly switch with fluorescent probes that bind to the active FtsZ conformation in filaments rather than to the free subunits, and label the division ring in bacterial cells. The FtsZ switch is important because it enables the filament treadmilling dynamics that in turn guides peptidoglycan synthesis during division. We have simultaneously developed cell-based methods to screen FtsZ inhibitors, which we aim to combine with competitive fluorescent assays to search for new antibiotic leads.
For more information, please see our research projects and collaborators (direct web address: http://www.cib.csic.es/tubulinas)
PC190723 GDP taxol GDP
Ligand binding cavities in FtsZ (left) and beta-tubulin (right)
TubZ from botulinum cs-t phage
Selected recent publications:
Huecas S et al. (2017) Self-organization of FtsZ polymers in solution reveals spacer role of the disordered C-terminal tail. Biophysical Journal, 113, 1831-1844 & cover
Wagstaff JM et al. (2017) A polymerysation-associated structural switch in FtsZ that enables treadmilling of model filaments. mBio 8, e00254-17
Artola M et al (2016) The structural assembly switch of cell división protein FtsZ probed with fluorescent allosteric inhibitors. Chemical Science, 2017, 8, 1525-1534. DOI: 10.1039/C6SC03792E
Araujo-Bazán L et al (2016) Cytological profile of antibacterial FtsZ inhibitors and synthetic peptide MciZ. Front. Microbiol. 7, 1558
Huecas S et al (2015) Beyond a fluorescent probe: inhibition of cell división protein FtsZ by mant-GTP elucidated by NMR and biochemical approaches. ACS Chem. Biol. 10, 834-843.
Ramirez-Aportela E et al., (2014) FtsZ filament dynamics and assembly switch unraveled with large-scale atomistic simulations. Biophys J 107,2164-2175
Prota A et al., (2014) A new tubulin binding site and pharmacophore for clinically relevant microtubule-destabilizing agents Proc. Natl. Acad. Sci. USA 111, 13817-13821
Pera B et al., (2013) New Interfacial Microtubule inhibitors of marine origin with Potent Antitumor Activity and a Distinct Mechanism. ACS Chem. Biol. 8, 2084-2094
Ruiz-Avila LB et al. (2013) Synthetic inhibitors of bacterial cell division targeting the GTP-binding site of FtsZ. ACS Chem. Biol. 8, 2072-2083.
Marcelo F et al. (2013) Interactions of bacterial cell division protein FtsZ with C8-substituted guanine nucleotide inhibitors. A combined NMR, biochemical and molecular modeling perspective. J. Am. Chem. Soc.135,16418-16428
Oliva MA et al. (2012) A TubZ tubulin homolog in a phage-encoded partition system. Proc. Natl. Acad. Sci. USA 109, 7711-7716
Artola, M., Ruíz-Avila, L.B., Ramírez-Aportela, E., Martínez, R.F., Araujo-Bazán, L., Vázquez-Villa, H., Martín-Fontecha, M., Oliva, M.A., Martín-Galiano, A.J., Chacón, P., López-Rodríguez, M.L., Andreu, J.M., Huecas, S. . The structural assembly switch of cell division protein FtsZ probed with fluorescent allosteric inhibitors. Chemical Science. 8:1525-1534.
Araújo-Bazán, L., Ruiz-Avila, L.B., Andreu, D., Huecas, S., Andreu, J.M. . Cytological profile of antibacterial FtsZ inhibitors and synthetic peptide MciZ. Frontiers in Microbiology. 7:-.
Jornet, D., Bosca, F., Andreu, J.M., Domingo, L.R., Tormos, R., Miranda, M.A. . Analysis of mebendazole binding to its target biomolecule by laser flash photolysis. Journal of Photochemistry and Photobiology B: Biology. 155:1-6.
Boscá, F., Sastre, G., Andreu, J.M., Jornet, D., Tormos, R., Miranda, M.A. . Drug-tubulin interactions interrogated by transient absorption spectroscopy. RSC Advances. 5:49451-49458.
Huecas, S., Marcelo, F., Perona, A., Ruiz-Ávila, L.B., Morreale, A., Canada, F.J., Jiménez-Barbero, J., Andreu, J.M. . Beyond a Fluorescent Probe: Inhibition of Cell Division Protein FtsZ by mant-GTP Elucidated by NMR and Biochemical Approaches. ACS Chemical Biology. 10:2382-2392.
Artola, M., Ruiz-Avila, L.B., Vergonos, A., Huecas, S., Araujo-Bazán, L., Martin-Fontecha, M., Vázquez-Villa, H., Turrado, C., Ramirez-Aportela, E., Hoegl, A., Nodwell, M., Barasoain, I., Chacon, P., Sieber, S.A., Andreu, J.M., Lopez-Rodriguez, M.L. . Effective GTP-replacing FtsZ inhibitors and antibacterial mechanism of action. ACS Chemical Biology. 10:834-843.
Martínez-Díez, M., Guillén-Navarro, M.J., Pera, B., Bouchet, B.P., Martínez-Leal, J.F., Barasoain, I., Cuevas, C., Andreu, J.M., García-Fernández, L.F., Díaz, J.F., Avilés, P., Galmarini, C.M. . PM060184, a new tubulin binding agent with potent antitumor activity including P-glycoprotein over-expressing tumors. Biochemical Pharmacology. 88:291-302.
Prota, A.E., Bargsten, K., Diaz, J.F., Marsh, M., Cuevas, C., Liniger, M., Neuhaus, C., Andreu, J.M., Altmann, K.-H., Steinmetz, M.O. . A new tubulin-binding site and pharmacophore for microtubule-destabilizing anticancer drugs. Proceedings of the National Academy of Sciences of the United States of America. 111:13817-13821.
Ramírez-Aportela, E., López-Blanco, J.R., Andreu, J.M., Chacón, P. . Understanding nucleotide-regulated FtsZ filament dynamics and the monomer assembly switch with large-scale atomistic simulations. Biophysical Journal. 107:2164-2176.
-Discovery and validation of therapeutic targets CM S2010/BMD-2353 (2012-2016)
-Molecular basis of segrosome organization in type III partition systems. BFU2013-47014-P (2014-2017)
-Targeting bacterial cell division protein FtsZ with small molecules and fluorescent probes. BFU2014-51823-R (2015-2018).