Molecular mechanism for functional interaction between DnaA protein and acidic phospholipids - Identification of important amino acids

DnaA protein, the initiator of chromosomal DNA replication in Escherichia c oli, seems to be reactivated hom the ADP-bound form to its ATP-bound form t hrough stimulation of ADP release by acidic phospholipids such as cardiolip in; We previously reported that two potential amphipathic helixes (Lys-327 to Ile-344 and Asp-357 to Val-374) of DnaA protein are involved in the func tional interaction between DnaA and cardiolipin, In relation to one of thes e helixes (Asp-357 to Val-374), we demonstrated that basic amino acids in t he helix, especially Lys-372, are vital for this interaction. In this study , we have identified an amino acid in the second potential amphipathic heli x (Lys-327 to Ile-344), which would also appear to be involved in the inter action. We constructed three mutant dnaA genes with a single mutation (dnaA R328E, dnaAR334E, and dnaAR342E) and examined the function of the mutant pr oteins. DnaAR328E, but not DnaAR334E and DnaAR342E, was found to be more re sistant to inhibition of its ATP binding activity by cardiolipin than the w ild-type protein, The stimulation of ADP release from DnaAR328E by cardioli pin was also weaker than that observed with the other mutants and the wild- type protein, These results suggest that Arg-328 of DnaA protein is involve d in the functional interaction of this protein with acidic phospholipids. We propose that acidic phospholipids bind to two basic amino acid residues (Arg-328 and Lys-372) of DnaA protein and change the higher order structure of its ATP-binding pocket, which in turn stimulates the release of ADP fro m the protein.