TrwD, the Hexameric Traffic ATPase Encoded by Plasmid R388, Induces Membrane Destabilization and Hemifusion of Lipid Vesicles

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Abstract
TrwD, a hexameric ATP hydrolase encoded by plasmid R388, is a member of the PulE/VirB11 protein superfamily of traffic ATPases. It is essential for plasmid conjugation, particularly for expression of the conjugative W pilus. In the present study, we analyzed the effects that TrwD produced on unilamellar vesicles consisting of cardiolipin and phosphatidylcholine in equimolar amounts. TrwD induced dose-dependent vesicle aggregation and intervesicular mixing of the lipids located in the outer monolayers in the presence of calcium. It also induced extensive leakage of the vesicular aqueous contents. A point mutant of TrwD with a mutation in the P loop of the nucleotide-binding region (K203Q) that lacks both ATPase activity and the ability to support conjugation showed the same behavior as native TrwD in all of these processes, which were independent of the presence of ATP. Structure prediction methods revealed a close similarity to Helicobacter pylori protein HP0525, another member of the PulE/VirB11 family, whose crystal structure is known. The interpretation of our data in the light of this structure is that TrwD interacts with the lipid bilayer through hydrophobic regions in its N-terminal domain, which leads to a certain degree of membrane destabilization. TrwD appears to be a part of the conjugation machinery that interacts with the membranous systems in order to facilitate DNA transfer in bacteria.