title: Interactions of nucleotide cofactors with the Escherichia coli replication factor DnaC protein.
abstract: Quantitative analyses of the interactions of nucleotide cofactors with the Escherichia coli replicative factor DnaC protein have been performed using thermodynamically rigorous fluorescence titration techniques. This approach allowed us to obtain stoichiometries of the formed complexes and interaction parameters, without any assumptions about the relationship between the observed signal and the degree of binding. The stoichiometry of the DnaC-nucleotide complex has been determined in direct binding experiments with fluorescent nucleotide analogues, MANT-ATP and MANT-ADP. The stoichiometry of the DnaC complexes with unmodified ATP and ADP has been determined using the macromolecular competition titration method (MCT). The obtained results established that at saturation the DnaC protein binds a single nucleotide molecule per protein monomer. Analyses of the binding of fluorescent analogues and unmodified nucleotides to the DnaC protein show that ATP and ADP have the same affinities for the nucleotide-binding site, albeit the corresponding complexes have different structures, specifically affected by the presence of magnesium cations in solution. Although the presence of the gamma-phosphate does not affect the affinity, the structure of the triphosphate group is critical. While the affinity of ATP-gamma-S is the same as the affinity of ATP, the affinities of AMP-PNP and AMP-PCP are approximately 2 and approximately 4 orders lower than that of ATP, respectively. Moreover, the ribose plays a significant role in forming a stable complex. The binding constants of dATP and dADP are approximately 2 orders of magnitude lower than those for ribose nucleotides. The nucleotide-binding site of the DnaC protein is highly base specific. The intrinsic affinity of adenosine triphosphates and diphosphates is at least 3-4 orders of magnitude higher than for any of the other examined nucleotides. The obtained data indicate that the recognition mechanism of the nucleotide by the structural elements of the binding site is complex with the base providing the specificity and the ribose, as well as the second phosphate group contributing to the affinity. The significance of the results for the functioning of the DnaC protein is discussed.
authors.affiliation: Department of Human Biological Chemistry & Genetics, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1053, USA.
authors.forename: R
authors.initials: R
authors.lastname: Galletto
authors.forename: S
authors.initials: S
authors.lastname: Rajendran
authors.forename: W
authors.initials: W
authors.lastname: Bujalowski
mesh_terms: D000241:Adenosine / Q000737:chemistry; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000345:Affinity Labels / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001667:Binding, Competitive; D001965:Bromides / Q000737:chemistry; D004261:DNA Replication; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes / Q000737:chemistry; D046911:Macromolecular Substances; D015636:Magnesium Chloride / Q000737:chemistry; D009711:Nucleotides / Q000737:chemistry* / Q000378:metabolism*; D012266:Ribose / Q000737:chemistry; D012965:Sodium Chloride / Q000737:chemistry; D017670:Sodium Compounds / Q000737:chemistry; D013403:Sugar Phosphates / Q000737:chemistry; D062367:ortho-Aminobenzoates / Q000737:chemistry
chemical_list: D000345:Affinity Labels; D001426:Bacterial Proteins; D001965:Bromides; C071702:DnaC protein, E coli; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D009711:Nucleotides; D017670:Sodium Compounds; D013403:Sugar Phosphates; D062367:ortho-Aminobenzoates; D015636:Magnesium Chloride; D012965:Sodium Chloride; D000244:Adenosine Diphosphate; D012266:Ribose; C081523:3'-O-(N-methylanthraniloyl)adenosine 5'-diphosphate; C045473:3'-O-(N-methylanthraniloyl) ATP; D000255:Adenosine Triphosphate; D000241:Adenosine; C027938:sodium bromide