title: 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.
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.
authors: Department of Biochemistry and Molecular Biology, Department of Obstetrics and Gynecology, The Sealy Center for Structural Biology, and The Sealy Center for Cancer Cell Biology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1053, United States.
authors: Michal R
authors: MR
authors: Szymanski
authors: Paul J
authors: PJ
authors: Bujalowski
authors: Maria J
authors: MJ
authors: Jezewska
authors: Aleksandra M
authors: AM
authors: Gmyrek
authors: Wlodzimierz
authors: W
authors: Bujalowski
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000494:Allosteric Regulation; D001665:Binding Sites; D004265:DNA Helicases / Q000737:chemistry* / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013816:Thermodynamics
chemical_list: D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D009711:Nucleotides; C022571:adenosine 5'-O-(3-thiotriphosphate); D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C506743:priA protein, E coli; D004265:DNA Helicases