Determination of the secondary structure in solution of the Escherichia coli DnaA DNA-binding domain.
DnaA protein binds specifically to a group of binding sites collectively called as DnaA boxes within the bacterial replication origin to induce local unwinding of duplex DNA. The DNA-binding domain of DnaA, domain IV, comprises the C-terminal 94 amino acid residues of the protein. We overproduced and purified a protein containing only this domain plus a methionine residue. This protein was stable as a monomer and maintained DnaA box-specific binding activity. We then analyzed its solution structure by CD spectrum and heteronuclear multi-dimensional NMR experiments. We established extensive assignments of the 1H, 13C, and 15N nuclei, and revealed by obtaining combined analyses of chemical shift index and NOE connectivities that DnaA domain IV contains six alpha-helices and no beta-sheets, consistent with results of CD analysis. Mutations known to reduce DnaA box-binding activity were specifically located in or near two of the alpha-helices. These findings indicate that the DNA-binding fold of DnaA domain IV is unique among origin-binding proteins.
Obita
Takayuki
T
Department of Immunology, Kyushu University Graduate School of Pharmaceutical Sciences, 3-1-1 Maidashi, Higashi-ku, 812-8582, Fukuoka, Japan.
Iwura
Takafumi
T
Su'etsugu
Masayuki
M
Yoshida
Yoichiro
Y
Tanaka
Yoshitsugu
Y
Katayama
Tsutomu
T
Ueda
Tadashi
T
Imoto
Taiji
T
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria