Sequence-related protein export NTPases encoded by the conjugative transfer region of RP4 and by the cag pathogenicity island of Helicobacter pylori share similar hexameric ring structures

RP4 TrbB, an essential component of the conjugative transfer apparatus of t he broad-host-range plasmid RP4, is a member of the PuIE protein superfamil y involved in multicomponent machineries transporting macromolecules across the bacterial envelope, PulE-like proteins share several well conserved mo tifs, most notable a nucleoside triphosphate binding motif (P-loop). Helico bacter pylori HP0525 also belongs to the PuIE superfamily and is encoded by the pathogenicity island cag, involved in the inflammatory response of inf ected gastric epithelial cells in mammals, The native molecular masses of T rbB and HP0525 as determined by gel filtration and glycerol gradient centri fugation suggested a homohexameric structure in the presence of ATP and Mg2 +. In the absence of nucleotides and bivalent cations, TrbB behaved as a te tramer whereas the hexameric state of HP0525 remained unaffected. Electron microscopy and image processing demonstrated that TrbB and HP0525 form ring -shaped complexes (diameter: 12 nm) with a central region (diameter: 3 nm) of low electron density when incubated in the presence of ATP and Mg2+, How ever, the TrbB average image appeared to be more elliptical with strong two fold rotational symmetry whereas HP0525 complexes are regular hexagons, Six well defined triangle-shaped areas of high electron density were distingui shable in both cases. Covalent crosslinking of TrbB suggests that the hexam eric ring is composed from a trimer of dimers, because only dimeric, tetram eric, and hexameric species were detectable. The toroidal structure of TrbB and HP0525 suggests that both proteins catalyze a repetitive process, most probably translocating a cognate substrate across the inner membrane.