Abstract
DnaA oligomerizes when bound to origins of chromosomal replication. Structural analysis of a truncated form of DnaA from Aquifex aeolicus has provided insight into crucial conformational differences within the AAA+ domain that are specific to the ATP- versus ADP- bound form of DnaA. In this study molecular docking of ATP and ADP onto Escherichia coli DnaA, modeled on the crystal structure of Aquifex aeolicus DnaA, reveals changes in the orientation of amino acid residues within or near the vicinity of the nucleotide-binding pocket. Upon limited proteolysis with trypsin or chymotrypsin ADP-DnaA, but not ATP-DnaA generated relatively stable proteolytic fragments of various sizes. Examined sites of limited protease susceptibility that differ between ATP-DnaA and ADP-DnaA largely reside in the amino terminal half of DnaA. The concentration of adenine nucleotide needed to induce conformational changes, as detected by these protease susceptibilities of DnaA, coincides with the conversion of an inactive bacterial origin recognition complex (bORC) to a replication efficient pre-replication complex (pre-RC) at the E. coli chromosomal origin of replication (oriC).
Highlights
Initiation of Escherichia coli chromosomal replication at a unique 245 base pair origin of replication is mediated by DnaA protein [1,2]
Domain I is followed by a flexible linker, domain II, that spans between amino acid residues 87–134 [3,4]
This study suggested different boundaries for domain II, spanning amino acid residues 79–135 [11]
Summary
Initiation of Escherichia coli chromosomal replication at a unique 245 base pair origin of replication (oriC) is mediated by DnaA protein [1,2]. The results from proteolytic cleavage analyses (Figures 2 and 3) suggest that levels of ATP higher than that needed to form a nucleotide-bound form, but approximating that found in cells, alter the conformation of DnaA protein. Digestion of ADP-DnaA and ATP-DnaA with trypsin and chymotrypsin in the presence of approximately physiological levels of AATP reveal nucleotide-dependent conformational changes within DnaA domains I–III (Table 1 and Figure 4). Our study here shows that a similar amount of adenine nucleotide is needed to induce detectable conformational differences in ADP-DnaA versus ATP-DnaA, and it correlates to the level of ATP required for the conversion of bORC to pre-RC and subsequent opening of duplex DNA. The possibility that the several micromolar to millimolar ATP concentration might be required for saturating more than one binding site present on DnaA protein, a function required for the conversion of bORC to pre-RC cannot be excluded and the mechanistic detail on these studies needs to be further elaborated
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