Crystal structure of the complex of the interaction domains of Escherichia coli DnaB helicase and DnaC helicase loader: structural basis implying a distortion-accumulation mechanism for the DnaB ring opening caused by DnaC binding.

Loading the bacterial replicative helicase DnaB onto DNA requires a specific loader protein, DnaC/DnaI, which creates the loading-competent state by opening the DnaB hexameric ring. To understand the molecular mechanism by which DnaC/DnaI opens the DnaB ring, we solved 3.1-Å co-crystal structure of the interaction domains of Escherichia coli DnaB-DnaC. The structure reveals that one N-terminal domain (NTD) of DnaC interacts with both the linker helix of a DnaB molecule and the C-terminal domain (CTD) of the adjacent DnaB molecule by forming a three α-helix bundle, which fixes the relative orientation of the two adjacent DnaB CTDs. The importance of the intermolecular interface in the crystal structure was supported by the mutational data of DnaB and DnaC. Based on the crystal structure and other available information on DnaB-DnaC structures, we constructed a molecular model of the hexameric DnaB CTDs bound by six DnaC NTDs. This model suggested that the binding of a DnaC would cause a distortion in the hexameric ring of DnaB. This distortion of the DnaB ring might accumulate by the binding of up to six DnaC molecules, resulting in the DnaB ring to open.

Inhibition of Klebsiella pneumoniae DnaB helicase by the flavonol galangin.

Klebsiella pneumoniae is a ubiquitous opportunistic pathogen that colonizes at the mucosal surfaces in humans and causes severe diseases. Many clinical strains of K. pneumoniae are highly resistant to antibiotics. Here, we used fluorescence quenching to show that the flavonols galangin, myricetin, quercetin, and kaempferol, bearing different numbers of hydroxyl substituent on the aromatic rings, may inhibit dNTP binding of the primary replicative DnaB helicase of K. pneumoniae (KpDnaB), an essential component of the cellular replication machinery critical for bacterial survival. The binding affinity of KpDnaB to dNTPs varies in the following order: dCTP ~ dGTP > dTTP > dATP. Addition of 10 µM galangin significantly decreased the binding ability of KpDnaB to dATP, whereas the binding affinity of KpDnaB to dGTP that was almost unaffected. Our analyses suggest that these flavonol compounds may be used in the development of new antibiotics that target K. pneumoniae and other bacteria.