STUDY ON RATIONAL DEVELOPMENT OF NOVEL ANTIBIOTICS TAGRETING DnaA DOMAIN IV

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To establish rational process for developing novel antibiotics that targets DnaA protein, we analyzed structure-function relationship of the DNA-binding domain of DnaA, DnaA domain IV(DAD-IV). First, we overproduced and purified a truncated DnaA protein that contained only the DAD-IV. We found that DAD-IV sustains specific affinity for DNA and secondary structure by gel-retardation assay and CD analysis. Then we determined the secondary structure in solution of DAD-IV using NMR. Determined structure suggested that DAD-IV contained a three helices-bundled type helix-turn-helix motif. In addition, NMR analysis suggested that interaction sites of DAD-IV with DNA. Finally, we determined crystal structure of a complex of DAD-IV with DNA using X-ray diffraction analysis. Determined structure by this well coincided with the data obtained by NMR and by other biochemical and genetic analysis. Candidates of antibiotics were searched by the method of virtual screening, based on the structure of DAD-IV/DNA complex.