Membrane regulation of the chromosomal replication activity of E. coli DnaA requires a discrete site on the protein.
The capacity of DnaA protein to initiate DNA synthesis at the chromosomal origin is influenced profoundly by the tightly bound nucleotides ATP and ADP. Acidic phospholipids can catalyze the conversion of inactive ADP-DnaA protein into the active ATP form. Proteolytic fragments of the nucleotide form of DnaA protein were examined to determine regions of the protein critical for functional interaction with membranes. A 35 kDa chymotryptic and 29 kDa tryptic fragment retained the tightly bound nucleotide. The fragments, whose amino-termini are within three residues of each other, but differ at their carboxyl ends, showed strikingly different behavior when treated with acidic phospholipids. The larger chymotryptic fragment released the bound nucleotide in the presence of acidic, but not neutral phospholipids. In contrast, the smaller tryptic fragment was inert to both forms of phospholipids. Acidic membranes, but not those composed of neutral phospholipids, protect from tryptic digestion a small portion of the segment that constitutes the difference between the 29 and 35 kDa fragments. The resulting 30 kDa tryptic fragment, which possesses this protected region, interacts functionally with acidic membranes to release the bound effector nucleotide. Inasmuch as the anionic ganglioside GM1, a compound structurally dissimilar to acidic glycerophospholipids, efficiently releases the nucleotide from DnaA protein, an acidic surface associated with a hydrophobic environment is the characteristic of the membrane that appears crucial for regulatory interaction with DnaA protein.
Garner
J
J
Department of Biochemistry and Molecular Biotechnology, Georgetown University Medical Center, Washington, DC 20007, USA.
Crooke
E
E
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002876:Chromosomes, Bacterial; D002918:Chymotrypsin / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D005732:Gangliosides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D010743:Phospholipids; D018741:Replication Origin
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D005732:Gangliosides; D010743:Phospholipids; D000255:Adenosine Triphosphate; D002918:Chymotrypsin