Beyond a Fluorescent Probe: Inhibition of Cell Division Protein FtsZ by mant-GTP Elucidated by NMR and Biochemical Approaches
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- 作者：Sonia Huecas ; Filipa Marcelo ; Almudena Perona ; Laura B. Ruiz-脕vila ; Antonio Morreale ; F. Javier Ca帽ada ; Jes煤s Jim茅nez-Barbero ; Jos茅 M. Andreu
- 刊名：ACS Chemical Biology
- 出版年：2015
- 出版时间：October 16, 2015
- 年：2015
- 卷：10
- 期：10
- 页码：2382-2392
- 全文大小：739K
- ISSN：1554-8937
文摘
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鈥? and C3鈥?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鈥?mant-GTP in two ribose puckering conformations and 3鈥?mant-GTP in the preferred C2鈥?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.