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. TrwBΔN70 contains the soluble domain of TrwB. It was constructed by deletion of trwB sequences containing TrwB N-proximal transmembrane segments. Purified TrwBΔN70 protein bound tightly the fluorescent ATP analogue TNP-ATP (K8 = 8.7 μM) but did not show measurable ATPase or GTPase activity. A single ATP 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 acid alteration in the ATP-binding signature (K136T) was transfer-deficient. TrwBΔN70 also bound DNA nonspecifically. DNA binding enhanced TrwC nic cleavage, providing the first evidence that directly links TrwB with conjugative DNA processing. Since DNA bound by TrwBΔN70 also showed increased negative superhelicity (as shown by increased sensitivity to topoisomerase I), nic cleavage enhancement was assumed to be a consequence of the increased single-stranded nature of DNA around nic. The mutant protein TrwB(K136T)ΔN70 was indistinguishable from TrwBΔN70 with respect to the above properties, indicating that TrwB ATP binding activity is not required for them. The reported properties of TrwB suggest potential functions for conjugative coupling proteins, both as triggers of conjugative DNA processing and as motors in the transport process.This work was supported by Dirección General de Investigación Científica y Técnica (Spain) Grant PB95-1269 (to F. C.), Comisión Interministerial de Ciencia y Tecnologı́a (Spain) Grant PB96/0171, Basque Government Grant EX-1998-28, and University of the Basque Country Grant UPV 042.310-G03/98 (to F. M. G.).The costs of publication of this article were defrayed in part by the payment of page charges.This work was supported by Direccio´n General de Investigación Científica y Técnica (Spain) Grant PB95-1269 (to F. C.), Comisión Interministerial de Ciencia y Tecnología (Spain) Grant PB96/0171, Basque Government Grant EX-1998-28, and University of the Basque Country Grant UPV 042.310-G03/98 (to F. M. G.).Peer Reviewe