An amphipathic alpha-helix including glutamates 509 and 516 is crucial formembrane translocation of adenylate cyclase toxin and modulates formation and cation selectivity of its membrane channels

The Bordetella pertussis adenylate cyclase toxin-hemolysin (ACT or CyaA) is a multifunctional protein. It forms small cation-selective channels in tar get cell and lipid bilayer membranes and it delivers into cell cytosol the amino-terminal adenylate cyclase (AC) domain, which catalyzes uncontrolled conversion of ATP to cAMP and causes cell intoxication. Here, we demonstrat e that membrane translocation of the AC domain into cells is selectively di ssociated from ACT membrane insertion and channel formation when a helix-br eaking proline residue is substituted for glutamate 509 (Glu-509) within a predicted transmembrane amphipathic alpha-helix. Neutral substitutions of G lu-509 had little effect on toxin activities. In contrast, charge reversal by lysine substitutions of the Glu-509 or of the adjacent Glu-516 residue r educed the capacity of the toxin to translocate the AC domain across membra ne and enhanced significantly its specific hemolytic activity and channel f orming capacity in lipid bilayer membranes. Combination of the E509K and E5 16K mutations in a single molecule further exacerbated hemolytic and channe l forming activity and ablated translocation of the AC domain into cells. T he lysine substitutions strongly decreased the cation selectivity of the ch annels, indicating that Glu-509 and Glu-516 are located within or close to the membrane channel. These results suggest that the structure including gl utamate residues 509 and 516 is critical for AC membrane translocation and channel forming activity of ACT.