Characterization of ATP and DNA binding activities of TrwB, the coupling protein essential in plasmid R388 conjugation

TrwB is the conjugative coupling protein of plasmid R388, TrwB Delta N70 co ntains the soluble domain of TrwB, It was constructed by deletion of trwB s equences containing TrwB N-proximal transmembrane segments. Purified TrwB D elta N70 protein bound tightly the fluorescent ATP analogue TNP-ATP (K-s = 8.7 mu M) but did not show measurable ATPase or GTPase activity. A single A TP binding site was found per TrwB monomer, An intact ATP-binding site was essential for R388 conjugation, since a TrwB mutant with a single amino aci d alteration in the ATP-binding signature (K136T) was transfer-deficient, T rwB Delta N70 also bound DNA nonspecifically. DNA binding enhanced TrwC nic cleavage, providing the first evidence that directly links TrwB with conju gative DNA processing. Since DNA bound by TrwB Delta N70 also showed increa sed negative superhelicity las shown by increased sensitivity to topoisomer ase I), nic cleavage enhancement was assumed to be a consequence of the inc reased single-stranded nature of DNA around nic, The mutant protein TrwB(K1 36T)Delta N70 was indistinguishable from TrwB Delta N70 with respect to the above properties, indicating that TrwB ATP binding activity is not require d for them. The reported properties of TrwB suggest potential functions for conjugative coupling proteins, both as triggers of conjugative DNA process ing and as motors in the transport process.