OLIGOMERIC STRUCTURE OF ESCHERICHIA-COLI PRIMARY REPLICATIVE HELICASEDNAB PROTEIN

The oligomeric structure of the Escherichia coli primary replicative h elicase DnaB protein in relation to the functions of the enzyme and th e energetics of its stability has been characterized. Sedimentation eq uilibrium, sedimentation velocity, and ligand binding studies show tha t, in solutions containing magnesium ions, the DnaB helicase exists as a stable hexamer over a wide protein concentration range (similar to 10(-7) to 10(-5) M (hexamer)). The sedimentation coefficient of the he xamer (s(20,w)(0) = 10.3 +/- 0.3 S) provides an apparent frictional ra tio of 1.09 +/- 0.03, which suggests that the hexamer has a nonspheric al shape and, when modeled as a prolate ellipsoid of revolution, has a n axial ratio of a/b = 2.6 +/- 0.6. Magnesium ions play a crucial stru ctural role in stabilizing the hexameric structure of the DnaB helicas e. In the absence of Mg2+, the DnaB protein forms a trimer that, at lo w protein concentrations, dissociates into monomers, Analysis of the s edimentation data indicates that the dimerization of the trimers into the active DnaB hexamer is accompanied by an uptake of similar to 4 ma gnesium cations. The sedimentation coefficient of the DnaB monomer (s( 20,w)(0) = 2.8 +/- 0.3 S) provides an apparent frictional ratio of 1.2 2 +/- 0.05, which indicates that the monomer has an elongated structur e with an axial ratio of a/b = 5.2 +/- 0.8 when modeled as a prolate e llipsoid of revolution. Analysis of the ratio of the sedimentation coe fficients (the sedimentation ratio) of the DnaB hexamer and monomer, w hich depends solely on the shape of the protomer and the mode of aggre gation, strongly suggests that elongated DnaB protomers aggregate with cyclic symmetry in which the protomer-protomer contacts are limited t o only two neighboring subunits.