Oligomeric structure of Escherichia coli primary replicative helicase DnaB protein.

Abstract

The oligomeric structure of the Escherichia coli primary replicative helicase DnaB protein in relation to the functions of the enzyme and the energetics of its stability has been characterized. Sedimentation equilibrium, sedimentation velocity, and ligand binding studies show that, in solutions containing magnesium ions, the DnaB helicase exists as a stable hexamer over a wide protein concentration range (approximately 10(-7) to 10(-5) M (hexamer)). The sedimentation coefficient of the hexamer (s0(20,w) = 10.3 +/- 0.3 S) provides an apparent frictional ratio of 1.09 +/- 0.03, which suggests that the hexamer has a nonspherical shape and, when modeled as a prolate ellipsoid of revolution, has an axial ratio of a/b = 2.6 +/- 0.6. Magnesium ions play a crucial structural role in stabilizing the hexameric structure of the DnaB helicase. In the absence of Mg2+, the DnaB protein forms a trimer that, at low protein concentrations, dissociates into monomers. Analysis of the sedimentation data indicates that the dimerization of the trimers into the active DnaB hexamer is accompanied by an uptake of approximately 4 magnesium cations. The sedimentation coefficient of the DnaB monomer (s0(20,w) = 2.8 +/- 0.3 S) provides an apparent frictional ratio of 1.22 +/- 0.05, which indicates that the monomer has an elongated structure with an axial ratio of a/b = 5.2 +/- 0.8 when modeled as a prolate ellipsoid of revolution. Analysis of the ratio of the sedimentation coefficients (the sedimentation ratio) of the DnaB hexamer and monomer, which depends solely on the shape of the protomer and the mode of aggregation, strongly suggests that elongated DnaB promoters aggregate with cyclic symmetry in which the protomer-protomer contacts are limited to only two neighboring subunits.