Identification of amino acids involved in the functional interaction between DnaA protein and acidic phospholipids

DnaA protein, the initiator of chromosomal DNA replication in Escherichia c oil, seems to be regulated through its binding to acidic phospholipids, suc h as cardiolipin. In our previous paper (Hase, M., Yoshimi, T,Ishikawa, Y., Ohba, A., Guo, L., Mima, S., Makise, M., Yamaguchi, Y., Tsuchiya, T., and Mizushima, T, (1998) J, Biol, Chem. 273, 28651-28656), we found that mutant DnaA protein (DnaA431), in which three basic amino acids (Arg(360), Arg(36 4) and Lys(372)) were mutated to acidic amino acids showed a decreased abil ity to interact with cardiolipin in vitro, suggesting that DnaA protein bin ds to cardiolipin through an ionic interaction. In this study, we construct three mutant dnaA genes each with a single mutation and examined the funct ion of the mutant proteins in vitro and in vivo. All mutant proteins mainta ined activities for DNA replication and ATP binding, A mutant protein in wh ich Lys(372) was mutated to Glu showed the weakest interaction with cardiol ipin among these three mutant proteins. Thus, Lys(372) seems to play an imp ortant role in the interaction between DnaA protein and acidic phospholipid s. Plasmid complementation analyses revealed that all these mutant proteins , including DnaA431 could function as an initiator for chromosomal DNA repl ication in vivo.