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

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.

The mating pair formation system of conjugative plasmids—A versatile secretion machinery for transfer of proteins and DNA

“…This may be the reason why nucleotide binding motif (Walker A) mutations of VirB11 fail to transfer the T-DNA substrate to the channel components VirB6 and VirB8, as detected by TrIP analysis in A. tumefaciens (Atmakuri et al, 2004). The Wnding that VirB11 induces membrane destabilization and hemifusion of lipid vesicles in vitro (Machón et al, 2002) furthermore suggests that VirB11 may function for facilitating membrane insertion of Mpf proteins that are part of the envelope-spanning Mpf complex. Analysis of the structure of VirB11 (HP0525) has indeed revealed a close structural similarity to other proteins exerting a function involving membrane fusion processes (see below).…”

Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery

“…While VirB4 together with VirB11 are required for pilus biogenesis and substrate translocation, VirD4 plays the role of a coupling protein, responsible for the recruitment of the substrate to the T4S system channel (Cabezon et al , 1997, 2015; Cascales & Christie, 2004). VirB11 has been reported as a hexamer in different systems (Machon et al , 2002; Savvides et al , 2003; Hare et al , 2006). VirD4 and VirB4, though expected to form hexamers, have been identified in monomeric and dimeric forms as well (Gomis‐Ruth et al , 2001; Schroder et al , 2002; Rabel et al , 2003; Arechaga et al , 2008; Mihajlovic et al , 2009; Durand et al , 2011; Pena et al , 2012; Wallden et al , 2012).…”

Conjugative Plasmid Transfer in Gram-Positive Bacteria

“…The crystal structure led to a model in which the VirB11-type ATPases function as GroEL-like chaperones in translocation of unfolded proteins across the cytoplasmic membrane (117,223). For the TrwD ATPase of R388, association with membrane vesicles that was independent of ATP hydrolysis was demonstrated, so that the protein could indeed act as a chaperone involved in the translocation of transfer components across the membranous system (131).…”