DnaA protein Lys-415 is close to the ATP-binding site: ATP-pyridoxal affinity labeling.
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 gamma-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.
Kubota
T
T
Department of Molecular Microbiology, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Ito
Y
Y
Sekimizu
K
K
Tagaya
M
M
Katayama
T
T
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D014357:Trypsin / Q000737:chemistry
D000345:Affinity Labels; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D029968:Escherichia coli Proteins; D010455:Peptides; C417675:pyridoxal 5'-triphosphoadenosine; D000255:Adenosine Triphosphate; D014357:Trypsin; D008239:Lysine