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Título: | Insights into nucleotide recognition by cell division protein FtsZ from a mant-GTP competition assay and molecular dynamics. |
Autor: | Schaffner-Barbero, Claudia CSIC; Gil-Redondo, Rubén; Ruiz-Avila, Laura B. CSIC; Huecas, Sonia CSIC ORCID ; Läppchen, Tilman; den Blaauwen, Tanneke; Díaz, José Fernando CSIC ORCID ; Morreale, Antonio; Andreu, José Manuel CSIC ORCID | Palabras clave: | Bacterial proteins Cell division protein FtsZ Binding sites Cytoskeletal proteins X-ray crystallography Molecular dynamics simulation Anthranilic acids Methanococcus Pseudomonas aeruginosa P. aeruginosa |
Fecha de publicación: | dic-2010 | Editor: | American Chemical Society | Citación: | Biochemistry, 49 (49): 10458–10472 (2010) | Resumen: | 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. | Descripción: | 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/bi101577p | Versión del editor: | http://pubs.acs.org/doi/full/10.1021/bi101577p http://dx.doi.org/10.1021/bi101577p |
URI: | http://hdl.handle.net/10261/42436 | DOI: | 10.1021/bi101577p | ISSN: | 0006-2960 | E-ISSN: | 1520-4995 |
Aparece en las colecciones: | (CIB) Artículos |
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