GLOBAL CONFORMATIONAL TRANSITIONS IN ESCHERICHIA-COLI PRIMARY REPLICATIVE HELICASE DNAB PROTEIN-INDUCED BY ATP, ADP, AND SINGLE-STRANDED-DNA BINDING - MULTIPLE CONFORMATIONAL, STATES OF THE HELICASE HEXAMER

The direct evidence of dramatic conformational changes of the DnaB hex amer, induced by nucleotide binding, and the presence of multiple conf ormational states of the enzyme have been obtained by using analytical sedimentation equilibrium, sedimentation velocity studies, and the ri gorous fluorescence titration technique. Equilibrium sedimentation mea surements show that in the presence of the ATP nonhydrolyzable analog, AMP-PNP, the DnaB helicase fully preserves its hexameric structure. H owever, in the presence of the saturating concentration of AMP-PNP, th e sedimentation coefficient of the hexamer is s(20,w) = 11.9 +/- 0.2 c ompared to the sedimentation coefficient s(20,w) = 10.5 +/- 0.2 of the free DnaB helicase hexamer. This large sedimentation coefficient chan ge indicates dramatic global conformational transitions of the hexamer , encompassing all six subunits, upon binding the ATP analog, In the p resence of ADP, the sedimentation coefficient is s(20,w) = 11.4 +/- 0. 2, indicating that the conformation of the ADP form of the hexamer is different from the ATP form. The sedimentation coefficient of the tern ary complex DnaB-(AMP-PNP)-d epsilon G(p epsilon A)(19), s(20,w) = 12. 4, suggests that the DnaB helicase undergoes further conformational ch anges upon binding single-stranded DNA (ssDNA). The large global struc tural changes correlate with the functional activities of the enzyme, In the absence of the ATP analog, the hexamer exists in a ''closed'' c onformation which has extremely low affinity toward ssDNA. Upon bindin g the ATP analog, the DnaB hexamer transforms into a ''tense'' state w hich binds ssDNA with an affinity of similar to 4 orders of magnitude higher than in the absence of the nucleotide, In the presence of ADP, the DnaB hexamer assumes a ''relaxed'' conformation. The functional di fference between these two conformations is reflected in the much weak er allosteric effect of ADP on the ssDNA binding with the affinity con stant similar to 3 orders of magnitude weaker than in the presence of the ATP analog (tense state).