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 me chanism, we used the affinity-labeling agent APT-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 co mpetitive mode. Binding stoichiometry was 0.28 ATP-pyridoxal/DnaA molecule, a value consistent with that of ATP. Thus, ATP-pyridoxal was a potent anta gonist for the DnaA ATP-binding site. The labeled DnaA protein was inactive for minichromosome replication in vitro, suggesting that conformation of t he region is important for DnaA activity. Isolation of the labeled, tryptic fragment and the Edman degradation revealed that ATP-pyridoxal modified Ly s-415. Thus, this residue is likely close to the bound ATP. Since Lys-415 i s located in the DNA-binding domain, these findings imply internal interact ion between the domains for ATP binding and DNA binding. (C) 2001 Academic Press.