Multiple global conformational states of the hexameric RepA helicase of plasmid RSF1010 with different ssDNA-binding capabilities are induced by different numbers of bound nucleotides. Analytical ultracentrifugation and dynamic light scattering studies.

Abstract

Global conformational transitions of the hexameric RepA helicase of plasmid RSF1010, induced by the nucleoside tri and di-phosphate binding, have been examined using analytical ultracentrifugation and dynamic light scattering techniques. The global structure of the RepA hexamer in solution, modeled as an oblate ellipsoid of revolution, is very different from its crystal structure, with the axial ratio of the ellipsoid being approximately 4.5 as compared to only approximately 2.4 in the crystal structure. The large axial ratio and the experimentally determined partial specific volume strongly suggest that, in solution, the diameter of the cross-channel of the hexamer is larger than approximately 17 A seen in the crystal. The global conformation of the helicase is modulated by a specific number of bound nucleotides. The enzyme exists in at least four conformational states, occurring sequentially as a function of the number of bound cofactors. These conformational states are different for ADP, as compared to beta,gamma-imidoadenosine 5'-triphosphate (AMP-PNP). Modulation of the global structure is separated into two phases, different for complexes with up to three bound nucleotides, from the effect observed at the saturating level of cofactors. This heterogeneity indicates different functional roles of the two modulation processes. Nucleotide control of helicase - single-stranded (ss)DNA interactions occurs through affecting the enzyme structure and the ssDNA affinity prior to DNA binding. Only one conformational state of the helicase, with two AMP-PNP molecules bound, has dramatically higher ssDNA-affinities than the complexes with ADP. Moreover the same state also has an increased site-size of the enzyme - ssDNA complexes. The implications of these findings for functional activities of a hexameric helicase are discussed.