Site-directed mutational analysis for the membrane binding of DnaA protein. 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 coli, interacts with acidic phospholipids, such as cardiolipin, and its activity seems to be regulated by membrane binding in cells. In this study we introduced site-directed mutations at the positions of hydrophobic or basic amino acids which are conserved among various bacteria species and which are located in the putative membrane-binding region of DnaA protein (from Asp357 to Val374). All mutant DnaA proteins showed much the same ATP and ADP binding activity as that of the wild-type protein. The release of ATP bound to the mutant DnaA protein, in which three hydrophobic amino acids were mutated to hydrophilic ones, was stimulated by cardiolipin, as in the case of the wild-type protein. On the other hand, the release of ATP bound to another mutant DnaA protein, in which three basic amino acids were mutated to acidic ones, was not stimulated by cardiolipin. These results suggest not only that the region is a membrane-binding domain of DnaA protein but also that these basic amino acids are important for the binding and the ionic interaction between the basic amino acids and acidic residues of cardiolipin and is involved in the interaction between DnaA protein and cardiolipin.
Hase
M
M
Faculty of Engineering, Oita University, Oita 870-1192, Japan.
Yoshimi
T
T
Ishikawa
Y
Y
Ohba
A
A
Guo
L
L
Mima
S
S
Makise
M
M
Yamaguchi
Y
Y
Tsuchiya
T
T
Mizushima
T
T
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding
D001426:Bacterial Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D010743:Phospholipids; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate