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

Masakazu Hase,Takeshi Yoshimi,Yuichi Ishikawa,Akiko Ohba,Lei Guo,Shinji Mima,Masaki Makise,Yoshihiro Yamaguchi,Tomofusa Tsuchiya,Tohru Mizushima

doi:10.1074/jbc.273.44.28651

Masakazu Hase, Takeshi Yoshimi + Show 8 more

Open Access

https://doi.org/10.1074/jbc.273.44.28651

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Abstract

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.

Highlights

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
These results suggest that adenine nucleotides bound to DnaA protein regulate the initiation of chromosomal DNA replication in E. coli cells
Site-directed Mutation for the Functional Interaction between Acidic Phospholipids and DnaA Protein—Since, in general, protein-lipid interactions are mediated by hydrophobic interactions between the hydrophobic moiety of lipids and hydrophobic amino acids in proteins, the amino acids most likely to be involved in the interaction of DnaA protein with phospholipids are hydrophobic ones

Summary

In order to couple the initiation of DNA replication with cell

Division, the initiator protein should be inactivated soon after the initiation of DNA replication to prevent overinitiation and it should be activated after an appropriate period for the replication cycle. As for the activation of DnaA protein for initiation of DNA replication, membrane phospholipids were reported to activate the inactive ADP-binding form of DnaA protein. In particular cardiolipin (CL), decrease the affinity of DnaA protein for adenine nucleotides [14,15,16,17], and activate the DnaA protein from the ADP-binding form to the ATP-binding form in the presence of high concentrations of ATP by stimulating the exchange of ADP with ATP [14]. We previously reported that artificial membranes consisting of a mixture of anionic and cationic phospholipids which mimic biological membranes can decrease the affinity of DnaA protein for adenine nucleotides under conditions in which acidic phospholipids form cluster structures [17]. A potential amphipathic helix (from Asp357 to Val374 of DnaA protein) located in this region may be a membrane-binding domain [1, 18]. We examined the effects of site-directed mutations on the membrane binding to DnaA protein

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION