Amino-terminal hydrophobic region of Helicobacter pylori vacuolating cytotoxin (VacA) mediates transmembrane protein dimerization

Helicobacter pylori VacA is a secreted protein toxin that forms channels in lipid bilayers and induces multiple structural and functional alterations in eukaryotic cells. A unique hydrophobic segment at the amino terminus of VacA contains three tandem repeats of a GxxxG motif that is characteristic of transmembrane dimerization sequences. To examine functional properties o f this region, we expressed and analyzed ToxR-VacA-maltose binding protein fusions using the TOXCAT system, which was recently developed by W. P. Russ and D. M. Engelman (Proc, Natl, Acad. Sci. USA 96:863-868, 1999) to study transmembrane helix-helix associations in a natural membrane environment. A wild-type VacA hydrophobic region mediated insertion of the fusion protein into the inner membrane of Escherichia coli and mediated protein dimerizat ion. A fusion protein containing a mutant VacA hydrophobic region tin which glycine 14 of VacA was replaced by alanine) also inserted into the Inner m embrane but dimerized significantly less efficiently than the fusion protei n containing the wild-type VacA sequence. Based on these results, we specul ate that the wild-type VacA amino-terminal hydrophobic region contributes t o oligomerization of the toxin within membranes of eukaryotic cells.