The E. coli replication factor DnaC protein exists in two conformations with different nucleotide binding capabilities. I. Determination of the binding mechanism using ATP and ADP fluorescent analogues

The kinetic mechanism of binding of ATP and ADP fluorescent analogues to the E. coli replicative factor DnaC protein has been studied using the fluorescence stopped-flow technique. The experiments have been performed under pseudo-first-order conditions with respect to the nucleotide cofactor or the DnaC concentration. Three relaxation processes are observed at a large excess of the nucleotide, while only two relaxation processes are detected in the excess of the protein. Such behavior of the kinetic system is a diagnostic indication of the presence of the protein conformational equilibrium prior to the ligand binding. The obtained data indicate that the minimum mechanism that describes the observed kinetics includes the conformational transition of the DnaC protein, prior to nucleotide binding, followed by the two-step, sequential association of the cofactor to only one of the protein conformations. In the examined solution conditions, the conformation of the DnaC protein is shifted toward the state (DnaC)₂ that binds the nucleotide. The lack of any cofactor binding to the (DnaC)₁ state points to the existence of a stringent locking mechanism of the nucleotide binding-site in the protein. Binding of ATP and ADP analogues obeys the same mechanism, with similar rate constants, indicating that ATP and ADP analogues bind to the same protein conformation. The (C)₁ intermediate dominates the distribution of the DnaC protein population in the presence of cofactors. The formation of (C)₁ is accompanied by a low nucleotide fluorescence increase, indicating a hydrophilic environment around the ribose of bound cofactors. Transition to (C)₂ places the ribose region in a highly hydrophobic environment with relative molar fluorescence intensity ∼8-fold higher than that of the free cofactor. The significance of these results for the functioning of the DnaC protein is discussed.