Determination of the secondary structure in solution of the Escherichia coli DnaA DNA-binding domain

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• 作者：Obita ; Takayuki ; Iwura ; Takafumi ; Su’ ; etsugu ; Masayuki ; Yoshida ; Yoichiro ; Tanaka ; Yoshitsugu ; et. al.
• 关键词：DnaA ; DNA-binding domain ; Circular dichroism ; NMR ; Escherichia coli ; DNA replication ; Origin-binding protein ; Secondary structure ; Chemical shift index ; Solution structure
• 刊名：Biochemical and Biophysical Research Communications
• 出版年：2002
• 出版时间：November 22, 2002
• 年：2002
• 卷：299
• 期：1
• 页码：42-48
• 全文大小：233 K

文摘

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 α-helices and no β-sheets, consistent with results of CD analysis. Mutations known to reduce DnaA box-binding activity were specifically located in or near two of the α-helices. These findings indicate that the DNA-binding fold of DnaA domain IV is unique among origin-binding proteins.