Characterization of the interaction between DNA gyrase inhibitor and DNA gyrase of Escherichia coli.
Escherichia coli DNA gyrase is comprised of two subunits, GyrA and GyrB. Previous studies have shown that GyrI, a regulatory factor of DNA gyrase activity, inhibits the supercoiling activity of DNA gyrase and that both overexpression and antisense expression of the gyrI gene suppress cell proliferation. Here we have analyzed the interaction of GyrI with DNA gyrase using two approaches. First, immunoprecipitation experiments revealed that GyrI interacts preferentially with the holoenzyme in an ATP-independent manner, although a weak interaction was also detected between GyrI and the individual GyrA and GyrB subunits. Second, surface plasmon resonance experiments indicated that GyrI binds to the gyrase holoenzyme with higher affinity than to either the GyrA or GyrB subunit alone. Unlike quinolone antibiotics, GyrI was not effective in stabilizing the cleavable complex consisting of gyrase and DNA. Further, we identified an 8-residue synthetic peptide, corresponding to amino acids (89)ITGGQYAV(96) of GyrI, which inhibits gyrase activity in an in vitro supercoiling assay. Surface plasmon resonance analysis of the ITGGQYAV-containing peptide-gyrase interaction indicated a high association constant for this interaction. These results suggest that amino acids 89--96 of GyrI are essential for its interaction with, and inhibition of, DNA gyrase.
Nakanishi
Akira
A
Cellular Physiology Laboratory, the Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
Imajoh-Ohmi
Shinobu
S
Hanaoka
Fumio
F
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D027081:DNA Gyrase / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D021122:Protein Subunits; D012097:Repressor Proteins; D020349:Surface Plasmon Resonance
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D021122:Protein Subunits; D012097:Repressor Proteins; C110533:SbmC protein, E coli; D027081:DNA Gyrase