Mutational analysis of conjugative coupling protein TrwB

Abstract

The coupling protein TrwB is a component of the conjugal machinery that is responsible for connecting the substrate (the DNA strand covalently linked to a pilot protein) to the T4SS during conjugative DNA transfer of plasmid R388. Biochemical and structural data suggest that TrwB uses energy released from ATP hydrolysis to pump DNA after export of the pilot protein through the T4SS, working as a molecular motor. The 3D structure of TrwBΔN70, the cytoplasmic domain of TrwB, revealed a hexameric structure similar to other molecular motor. Based on this 3D structure, a series of point mutants were constructed. We have analyzed in vivo and in vitro properties of TrwB mutants which were only transfer deficient in standard conjugation assays under TrwB limiting condition. We have focused on lysine residues which protrude into the internal channel of the TrwB hexamer because this is the candidate region for DNA interaction, since the transferred DNA is expected to travel through the interior of the hexamer. The results show that lysines protruding into the internal channel have a deregulated ATPase activity; the enhancing effect of ssDNA and TrwA in ATPase activity was also affected in these mutants.Peer Reviewe