Membrane-catalyzed Nucleotide Exchange on DnaA

Abstract

DnaA is the initiator protein for chromosomal replication in bacteria; its activity plays a central role in the timing of the primary initiations within the Escherichia coli cell cycle. A controlled, reversible conversion between the active ATP-DnaA and the inactive ADP forms modulates this activity. In a DNA-dependent manner, bound ATP is hydrolyzed to ADP. Acidic phospholipids with unsaturated fatty acids are capable of reactivating ADP-DnaA by promoting the release of the tightly bound ADP. The nucleotide dissociation kinetics, measured in the present study with the fluorescent derivative 3-O-(N-methylantraniloyl)-5-adenosine triphosphate, was dependent on the density of DnaA on the membrane in a cooperative manner: it increased 5-fold with decreased protein density. At all surface densities the nucleotide was completely released, presumably due to protein exchange on the membrane. Distinct temperature dependences and the effect of the crowding agent Ficoll suggest that two functional states of DnaA exist at high and low membrane occupancy, ascribed to local macromolecular crowding on the membrane surface. These novel phenomena are thought to play a major role in the mechanism regulating the initiation of chromosomal replication in bacteria.

Type IV traffic ATPase TrwD as molecular target to inhibit bacterial conjugation

“…The ATPase activity of this DNA replication initiator is regulated by acidic phospholipids. The presence of saturated or unsaturated fatty acids on these phospholipids regulates the ADP-ATP exchange in the protein (Aranovich et al , 2006). Based on this, we decided to investigate the effect of uFAs on the ATPases involved in bacterial conjugation.…”

Macromolecular Crowding at Membrane Interfaces: Adsorption and Alignment of Membrane Peptides

“…It was for example suggested, that the crowding-dependent kinetics of DnaA serves as a signal for the state of the membrane during the cell cycle. 18 Bacterial osmosensing and cell volume control are probably mediated by crowding. 16,17 Conclusion The partitioning of biomolecules at cellular membrane surfaces depends strongly on the amount and composition of previously adsorbed molecules and intermolecular interactions are not negligible.…”

“…16,17 The influence of surface molecular crowding has recently been shown for the chromosomal replication initiation protein of Escherichia coli (DnaA). 18 The correlation of the dissociation kinetics of the ATP-DnaA complex with the membrane occupancy of DnaA is proposed to be a triggering event in activation of DnaA during the cell cycle. Furthermore, intuitive models for antibiotic activity of membrane peptides assume cooperative behavior of the lipid-associated peptides at high peptide-to-lipid ratios.…”

Crosstalk between DnaA Protein, the Initiator of Escherichia coli Chromosomal Replication, and Acidic Phospholipids Present in Bacterial Membranes

“…In fact, two different functional states of DnaA protein exists at high and low membrane occupancy, which influences the release of nucleotide from protein [98]. Using the fluorescent ATP derivative MANT-ATP, it has been shown that crowding of DnaA protein on membrane could be induced by changes in temperature or the presence of Ficoll as a crowding agent [98]. …”