PMID:21888358
| Citation |
Szymanski, MR, Bujalowski, PJ, Jezewska, MJ, Gmyrek, AM and Bujalowski, W (2011) The N-terminal domain of the Escherichia coli PriA helicase contains both the DNA- and nucleotide-binding sites. Energetics of domain--DNA interactions and allosteric effect of the nucleotide cofactors. Biochemistry 50:9167-83 |
|---|---|
| Abstract |
Functional interactions of the Escherichia coli PriA helicase 181N-terminal domain with the DNA and nucleotide cofactors have been quantitatively examined. The isolated 181N-terminal domain forms a stable dimer in solution, most probably reflecting the involvement of the domain in specific cooperative interactions of the intact PriA protein--double-stranded DNA (dsDNA) complex. Only one monomer of the domain dimer binds the DNA; i.e., the dimer has one effective DNA-binding site. Although the total site size of the dimer--single-stranded DNA (ssDNA) complex is ~13 nucleotides, the DNA-binding subsite engages in direct interactions with approximately five nucleotides. A small number of interacting nucleotides indicates that the DNA-binding subsites of the PriA helicase, i.e., the strong subsite on the helicase domain and the weak subsite on the N-terminal domain, are spatially separated in the intact enzyme. Contrary to current views, the subsite has an only slight preference for the 3'-end OH group of the ssDNA and lacks any significant base specificity, although it has a significant dsDNA affinity. Unlike the intact helicase, the DNA-binding subsite of the isolated domain is in an open conformation, indicating the presence of the direct helicase domain--N-terminal domain interactions. The discovery that the 181N-terminal domain possesses a nucleotide-binding site places the allosteric, weak nucleotide-binding site of the intact PriA on the N-terminal domain. The specific effect of ADP on the domain DNA-binding subsite indicates that in the intact helicase, the bound ADP not only opens the DNA-binding subsite but also increases its intrinsic DNA affinity. |
| Links |
PubMed PMC3205976 Online version:10.1021/bi201100k |
| Keywords |
Adenosine Diphosphate/metabolism; Adenosine Triphosphate/analogs & derivatives; Adenosine Triphosphate/metabolism; Allosteric Regulation; Binding Sites; DNA Helicases/chemistry; DNA Helicases/metabolism; DNA, Bacterial/chemistry; DNA, Bacterial/metabolism; DNA, Single-Stranded/chemistry; DNA, Single-Stranded/metabolism; Escherichia coli/chemistry; Escherichia coli/enzymology; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/metabolism; Models, Molecular; Nucleic Acid Conformation; Nucleotides/metabolism; Protein Structure, Tertiary; Thermodynamics |
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