DnaA protein Lys-415 is close to the ATP-binding site: ATP-pyridoxal affinity labeling

Toshio Kubota, Yuji Ito, Kazuhisa Sekimizu, Mitsuo Tagaya, Tsutomu Katayama

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Binding of ATP, but not of ADP, activates Escherichia coli DnaA protein for replicational initiation of the chromosome. To elucidate this switching mechanism, we used the affinity-labeling agent ATP-pyridoxal, which forms a covalent bond with the Lys residue located at or near the γ-phosphate of ATP. ATP-pyridoxal inhibited the ATP binding for DnaA protein, with a competitive mode. Binding stoichiometry was 0.28 ATP-pyridoxal/DnaA molecule, a value consistent with that of ATP. Thus, ATP-pyridoxal was a potent antagonist for the DnaA ATP-binding site. The labeled DnaA protein was inactive for minichromosome replication in vitro, suggesting that conformation of the region is important for DnaA activity. Isolation of the labeled, tryptic fragment and the Edman degradation revealed that ATP-pyridoxal modified Lys-415. Thus, this residue is likely close to the bound ATP. Since Lys-415 is located in the DNA-binding domain, these findings imply internal interaction between the domains for ATP binding and DNA binding.

Original languageEnglish
Pages (from-to)1141-1148
Number of pages8
JournalBiochemical and Biophysical Research Communications
Volume288
Issue number5
DOIs
Publication statusPublished - Nov 16 2001

Fingerprint

Pyridoxal
Labeling
Adenosine Triphosphate
Binding Sites
Proteins
Covalent bonds
Escherichia coli Proteins
DNA
Chromosomes
Stoichiometry
Adenosine Diphosphate
Escherichia coli
Conformations

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

DnaA protein Lys-415 is close to the ATP-binding site : ATP-pyridoxal affinity labeling. / Kubota, Toshio; Ito, Yuji; Sekimizu, Kazuhisa; Tagaya, Mitsuo; Katayama, Tsutomu.

In: Biochemical and Biophysical Research Communications, Vol. 288, No. 5, 16.11.2001, p. 1141-1148.

Research output: Contribution to journalArticle

Kubota, Toshio ; Ito, Yuji ; Sekimizu, Kazuhisa ; Tagaya, Mitsuo ; Katayama, Tsutomu. / DnaA protein Lys-415 is close to the ATP-binding site : ATP-pyridoxal affinity labeling. In: Biochemical and Biophysical Research Communications. 2001 ; Vol. 288, No. 5. pp. 1141-1148.
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