Membrane-catalyzed nucleotide exchange on DnaA. Effect of surface molecular crowding.
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.
Aranovich
Alexander
A
Department of Life Sciences, Ben-Gurion University of the Negev, P. O. B. 653, Beer-Sheva 84105, Israel.
Gdalevsky
Garik Y
GY
Cohen-Luria
Rivka
R
Fishov
Itzhak
I
Parola
Abraham H
AH
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D001426:Bacterial Proteins / Q000378:metabolism / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D004926:Escherichia coli / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D008968:Molecular Conformation; D009711:Nucleotides / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry; D011485:Protein Binding; D013696:Temperature; D062367:ortho-Aminobenzoates / Q000494:pharmacology
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D005456:Fluorescent Dyes; D009711:Nucleotides; D010743:Phospholipids; D062367:ortho-Aminobenzoates; C045473:3'-O-(N-methylanthraniloyl) ATP; D000255:Adenosine Triphosphate