Insights into nucleotide recognition by cell division protein FtsZ from a mant-GTP competition assay and molecular dynamics.
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.
Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.
Claudia
C
Schaffner-Barbero
Rubén
R
Gil-Redondo
Laura B
LB
Ruiz-Avila
Sonia
S
Huecas
Tilman
T
Läppchen
Tanneke
T
den Blaauwen
J Fernando
JF
Diaz
Antonio
A
Morreale
Jose M
JM
Andreu
D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D018360:Crystallography, X-Ray; D003598:Cytoskeletal Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000737:chemistry* / Q000378:metabolism*; D017017:Methanococcus / Q000737:chemistry / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D062367:ortho-Aminobenzoates / Q000737:chemistry* / Q000378:metabolism*
D001426:Bacterial Proteins; D003598:Cytoskeletal Proteins; C071525:FtsZ protein, Bacteria; D062367:ortho-Aminobenzoates; C063981:2'(3')-O-(N-methyl)anthraniloylguanosine 5'-triphosphate; D006160:Guanosine Triphosphate