title: Beyond a Fluorescent Probe: Inhibition of Cell Division Protein FtsZ by mant-GTP Elucidated by NMR and Biochemical Approaches.
abstract: FtsZ is the organizer of cell division in most bacteria and a target in the quest for new antibiotics. FtsZ is a tubulin-like GTPase, in which the active site is completed at the interface with the next subunit in an assembled FtsZ filament. Fluorescent mant-GTP has been extensively used for competitive binding studies of nucleotide analogs and synthetic GTP-replacing inhibitors possessing antibacterial activity. However, its mode of binding and whether the mant tag interferes with FtsZ assembly function were unknown. Mant-GTP exists in equilibrium as a mixture of C2'- and C3'-substituted isomers. We have unraveled the molecular recognition process of mant-GTP by FtsZ monomers. Both isomers bind in the anti glycosidic bond conformation: 2'-mant-GTP in two ribose puckering conformations and 3'-mant-GTP in the preferred C2' endo conformation. In each case, the mant tag strongly interacts with FtsZ at an extension of the GTP binding site, which is also supported by molecular dynamics simulations. Importantly, mant-GTP binding induces archaeal FtsZ polymerization into inactive curved filaments that cannot hydrolyze the nucleotide, rather than straight GTP-hydrolyzing assemblies, and also inhibits normal assembly of FtsZ from the Gram-negative bacterium Escherichia coli but is hydrolyzed by FtsZ from Gram-positive Bacillus subtilis. Thus, the specific interactions provided by the fluorescent mant tag indicate a new way to search for synthetic FtsZ inhibitors that selectively suppress the cell division of bacterial pathogens.
authors.affiliation: Centro de Investigaciones Biológicas, CIB-CSIC , Ramiro de Maeztu 9, 28040 Madrid, Spain.
authors.forename: Sonia
authors.initials: S
authors.lastname: Huecas
authors.affiliation: Centro de Investigaciones Biológicas, CIB-CSIC , Ramiro de Maeztu 9, 28040 Madrid, Spain.|UCIBIO, REQUIMTE , Dept. de Química, Faculdade de Ciências e Tecnologia, UNL, 2829-516 Caparica, Portugal.
authors.forename: Filipa
authors.initials: F
authors.lastname: Marcelo
authors.affiliation: Unidad de Bioinformática, Centro de Biología Molecular Severo Ochoa, CBMSO-CSIC , Cantoblanco, 28049 Madrid, Spain.
authors.forename: Almudena
authors.initials: A
authors.lastname: Perona
authors.affiliation: Centro de Investigaciones Biológicas, CIB-CSIC , Ramiro de Maeztu 9, 28040 Madrid, Spain.
authors.forename: Laura B
authors.initials: LB
authors.lastname: Ruiz-Ávila
authors.affiliation: Unidad de Bioinformática, Centro de Biología Molecular Severo Ochoa, CBMSO-CSIC , Cantoblanco, 28049 Madrid, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Morreale
authors.affiliation: Centro de Investigaciones Biológicas, CIB-CSIC , Ramiro de Maeztu 9, 28040 Madrid, Spain.
authors.forename: F Javier
authors.initials: FJ
authors.lastname: Cañada
authors.affiliation: Centro de Investigaciones Biológicas, CIB-CSIC , Ramiro de Maeztu 9, 28040 Madrid, Spain.
authors.forename: Jesús
authors.initials: J
authors.lastname: Jiménez-Barbero
authors.affiliation: Centro de Investigaciones Biológicas, CIB-CSIC , Ramiro de Maeztu 9, 28040 Madrid, Spain.
authors.forename: José M
authors.initials: JM
authors.lastname: Andreu
mesh_terms: D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D003598:Cytoskeletal Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006160:Guanosine Triphosphate / Q000737:chemistry* / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D008954:Models, Biological; D056004:Molecular Dynamics Simulation; D015394:Molecular Structure; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation; D062367:ortho-Aminobenzoates / Q000737:chemistry* / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D003598:Cytoskeletal Proteins; D005456:Fluorescent Dyes; C071525:FtsZ protein, Bacteria; D062367:ortho-Aminobenzoates; C063981:2'(3')-O-(N-methyl)anthraniloylguanosine 5'-triphosphate; D006160:Guanosine Triphosphate