The interaction of VacA with membranes involves: (i) a low pH activation that induces VacA monomerization in solution, (ii) binding of the monomers to the membrane, (iii) oligomerization and (iv) channel formation. To better understand the structure-activity relationship of VacA, we determined its topology in a lipid membrane by a combination of proteolytic, structural and fluorescence techniques. Residues 40-66, 111-169, 205-266, 548-574 and 723-767 were protected from proteolysis because of their interaction with the membrane. This last peptide was shown to most probably adopt a surface orientation. Both alpha-helices and beta-sheets were found in the structure of the protected peptides.