GTP hydrolysis of cell division protein FtsZ: evidence that the active site is formed by the association of monomers.
The essential prokaryotic cell division protein FtsZ is a tubulin homologue that forms a ring at the division site. FtsZ forms polymers in a GTP-dependent manner. Recent biochemical evidence has shown that FtsZ forms multimeric structures in vitro and in vivo and functions as a self-activating GTPase. Structural analysis of FtsZ points to an important role for the highly conserved tubulin-like loop 7 (T7-loop) in the self-activation of GTP hydrolysis. The T7-loop was postulated to form the active site together with the nucleotide-binding site on an adjacent FtsZ monomer. To characterize the role of the T7-loop of Escherichia coli FtsZ, we have mutagenized residues M206, N207, D209, D212, and R214. All the mutant proteins, except the R214 mutant, are severely affected in polymerization and GTP hydrolysis. Charged residues D209 and D212 cannot be substituted with a glutamate residue. All mutants interact with wild-type FtsZ in vitro, indicating that the T7-loop mutations do not abolish FtsZ self-association. Strikingly, in mixtures of wild-type and mutant proteins, most mutants are capable of inhibiting wild-type GTP hydrolysis. We conclude that the T7-loop is part of the active site for GTP hydrolysis, formed by the association of two FtsZ monomers.
Scheffers
Dirk-Jan
DJ
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
de Wit
Janny G
JG
den Blaauwen
Tanneke
T
Driessen
Arnold J M
AJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D003598:Cytoskeletal Proteins; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology; D020558:GTP Phosphohydrolases / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D008027:Light; D008274:Magnesium / Q000494:pharmacology; D008854:Microscopy, Electron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D001426:Bacterial Proteins; D003598:Cytoskeletal Proteins; C071525:FtsZ protein, Bacteria; D018698:Glutamic Acid; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D008274:Magnesium; D003545:Cysteine; D002118:Calcium