Beyond a Fluorescent Probe: Inhibition of Cell Division Protein FtsZ by <i>mant</i>-GTP Elucidated by NMR and Biochemical Approaches

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 <i>mant</i>-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 <i>mant</i> tag interferes with FtsZ assembly function were unknown. <i>Mant</i>-GTP exists in equilibrium as a mixture of C2′- and C3′-substituted isomers. We have unraveled the molecular recognition process of <i>mant</i>-GTP by FtsZ monomers. Both isomers bind in the <i>anti</i> glycosidic bond conformation: 2′-<i>mant</i>-GTP in two ribose puckering conformations and 3′-<i>mant</i>-GTP in the preferred C2′ endo conformation. In each case, the <i>mant</i> tag strongly interacts with FtsZ at an extension of the GTP binding site, which is also supported by molecular dynamics simulations. Importantly, <i>mant</i>-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 <i>Escherichia coli</i> but is hydrolyzed by FtsZ from Gram-positive <i>Bacillus subtilis</i>. Thus, the specific interactions provided by the fluorescent <i>mant</i> tag indicate a new way to search for synthetic FtsZ inhibitors that selectively suppress the cell division of bacterial pathogens