TraG-like proteins contribute in an essential manner to the translocation of protein and DNA substrates in conjugation and the related type IV secretion systems. Despite their significance for these processes, the nature of their activity remains obscure. To provide insights into the function of TraG-like proteins, biochemical properties of three members of this family originating from different type IV secretion systems, TraG, TraD, and HP0524, were determined. These proteins belong to conjugative DNA transfer systems of plasmids RP4 and F and to the pathogenicity-related type IV secretion system of gastric pathogen H. pylori
, respectively. TraG and TraD were purified as His-tagged full-length proteins (His6
-TraG and His6
-TraD), and HP0524 was purified as a truncated derivative lacking the predicted transmembrane domain (His6
-HP0524Δ1) (Fig. ). A pronounced tendency to form oligomers or aggregates was observed for each of these proteins (Fig. and Table ). TraG, TraD, and HP0524Δ1 were found to bind to DNA without sequence specificity (Fig. and ) and did not possess an intrinsic NTPase activity under the conditions tested (Fig. ). Specific and strong binding of RP4 TraG to relaxase TraI was demonstrated by SPR (Fig. ). Functionally essential domains and the membrane topology of TraG were determined by analysis of in-frame insertion mutants (Fig. and ). Thus, TraG was shown to be a membrane-anchored protein with a topology corresponding to that of its F and Ti plasmid analogues, TraD and VirD4 (15
The crystal structure of a truncated derivative of the TraG-like protein of plasmid R388 lacking the transmembrane part (TrwBΔN70) has recently been solved (22
). TrwB is proposed to form a hexameric, pore-like structure, strongly resembling the structure of F1
ATPase and ring helicases. The identification of functionally essential regions of TraG enables comparison with the related structural domains of TrwB. The core of the TrwB structure, consisting of the NBD, is apparently conserved in TraG (Fig. ) and is critical for TraG activity in mating experiments (4
) (Table ).
Taken together, the biochemical characteristics of the three TraG-like proteins presented in this study and the properties of TrwBΔN70 published earlier support the view that TraG-like proteins assemble as multimeric forms that are anchored in the inner membrane of the gram-negative bacterial cell envelope. Genetic and biochemical evidence clearly indicates a physical association between TraG-like proteins and components of the relaxosome via protein-protein and protein-DNA interactions. These findings lead to the hypothesis that TraG-like proteins form an inner membrane pore, which is specifically recognized by the secreted substrates. A model illustrating the proposed architecture of the TraG-associated RP4 relaxosome is shown (Fig. ). TraG is depicted as a homohexamer based on the structural data for TrwBΔN70 (22
). The relaxosome consists of relaxase TraI, which cleaves the T strand of oriT
DNA at the nic
site, along with accessory proteins contributing to relaxase activity (TraJ), relaxosome stability (TraH), and oriT
accessibility (TraK) (41
). Here, we propose that this complex is linked to the potential TraG inner membrane pore via TraG-TraI and TraG-DNA interactions.
FIG. 10. Proposed model for the RP4 relaxosome. TraG (red) is a membrane-anchored, multimeric protein probably forming a pore-like structure that could serve as a channel for translocation of the transferred ssDNA (T-DNA). The relaxase TraI (green) and the plasmid (more ...)
While this overall assembly is well supported by the present data, the role of TraG-like proteins in the macromolecular transport process remains speculative. The interaction of TraG-like proteins with DNA and with proteins that prepare substrate DNA for transfer implies a direct role for these proteins in the translocation mechanism itself or in its regulation. The inner membrane localization and postulated multimeric pore-like structure of the TraG-like proteins are highly suggestive of a translocation portal or passageway for exported substrates. Less consistent with this view is the observation that TraG-like proteins do not seem to exhibit NTP-hydrolyzing activity under the applied conditions (reference 37
and this work). The possibility that an accessory factor (or several factors) may be required for these proteins to function as active NTPases cannot be excluded.
Genetic analysis has revealed that hp0524
is absolutely required for infectivity of H. pylori
). Here, comparison of the biochemical properties of HP0524Δ1 to those of other TraG-like proteins of conjugative transfer systems revealed close similarities. In view of the fact that the 145-kDa CagA protein is the only known substrate for the type IV secretion system of H. pylori
, our finding that HP0524Δ1 binds to DNA is remarkable. A conjugation-like mechanism for DNA transfer between Helicobacter
strains was suggested earlier (27
), although the genetic determinants have not been identified. Two predicted relaxases encoded by open reading frames hp0996
) are possible candidates for interaction with HP0524. Thus, involvement of HP0524 in a DNA transfer system cannot be excluded. On the other hand, type IV secretion systems of pathogens have most probably evolved from conjugative DNA transfer systems (62
). Thus the DNA binding activity of HP0524 may be a residual activity from a TraG ancestor of a conjugative transfer system. Whether DNA is still actively transported by this secretion system or whether it is even involved in pathogenicity remains to be elucidated.
In type IV secretion systems, the transported substrates consist of a protein (such as CagA of H. pylori cag
) and/or of a protein complexed to DNA (such as TraI-oriT
of RP4). The A. tumefaciens
Ti plasmid secretion system transports the VirD2-T-DNA complex, along with virulence-associated proteins VirE2 and VirF, into plant cells. VirE2 and VirF translocation depends on the VirB/VirD4 transport system (related to the Mpf/TraG system of RP4) but does not require DNA transfer (58
). Secretion of CagA by H. pylori
is equally dependent on the corresponding VirB/VirD4 system of cag
). It is conceivable that analogous Mpf/TraG-dependent protein secretion exists in the conjugative transfer system of RP4. TraC, a functional analogue of VirE2, is transferred into recipient cells during RP4-mediated conjugation (45
). Since TraC and VirE2 are cytoplasmic proteins lacking a signal sequence for secretion by the sec
system (GSP system), TraG and VirD4 may possibly mediate their transport through the inner membrane.
The putative translocation activity of TraG-like proteins appears to be limited to crossing the inner membrane. It is notable that the only type IV secretion system clearly lacking a TraG homologue is found in B. pertussis
. The pertussis toxin liberation system, Ptl, employs a two-step mechanism for secretion. Pertussis toxin subunits rely on the sec
system for translocation through the inner membrane, and the Ptl system is responsible for transition of the holotoxin across the outer membrane barrier (8
). The activity of the Ptl transport system is limited to the outer membrane and is therefore distinct from that of the VirB prototype of type IV transporters, which convey substrates across both the inner and outer membranes. The notable absence of a traG
homologue in B. pertussis
implies that the putative ancestral TraG-like protein of this secretion system has been replaced by the sec
system of the host during evolution.
In type IV secretion systems the functional connection between TraG-like proteins and the envelope-spanning Mpf components remains uncertain. Physical interactions between the TraG-like proteins and Mpf proteins have been postulated (10
), but biochemical evidence for this remains to be supplied. The periplasmic domain of TraG could possibly mediate interactions of this type. During the present study we tested the possibility that TrbB, a membrane-associated cytosolic ATPase of the Mpf system of RP4 (31
), might provide an interface with TraG. However, a TrbB-TraG interaction was not detected by affinity chromatography of TrbB on a His6
-TraG-loaded Ni-NTA column, whereas the TraI-TraG interaction was detected by the same technique (25
). The most promising approaches for the detection of protein-protein interactions, SPR and affinity chromatography, are hampered by the limited solubility of Mpf proteins. Thus, direct evidence for a linkage between TraG-like proteins and components of the Mpf transfer machinery is still lacking. Resolution of this challenging aspect of TraG function will probably prove decisive to unraveling the mechanism of type IV secretion.