An amphipathic alpha-helix including glutamates 509 and 516 is crucial for membrane 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 target 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 demonstrate that membrane translocation of the AC domain into cells is selectively dissociated from ACT membrane insertion and channel formation when a helix-breaking proline residue is substituted for glutamate 509 (Glu-509) within a predicted transmembrane amphipathic alpha-helix. Neutral substitutions of Glu-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 reduced the capacity of the toxin to translocate the AC domain across membrane and enhanced significantly its specific hemolytic activity and channel forming capacity in lipid bilayer membranes. Combination of the E509K and E516K mutations in a single molecule further exacerbated hemolytic and channel forming activity and ablated translocation of the AC domain into cells. The lysine substitutions strongly decreased the cation selectivity of the channels, indicating that Glu-509 and Glu-516 are located within or close to the membrane channel. These results suggest that the structure including glutamate residues 509 and 516 is critical for AC membrane translocation and channel forming activity of ACT.
Osicková
A
A
Cellular Division, Institute of Microbiology of the Academy of Sciences of the Czech Republic, Vídeòská 1083, CZ-142 20 Prague 4, Czech Republic.
Osicka
R
R
Maier
E
E
Benz
R
R
Sebo
P
P
D037361:Adenylate Cyclase Toxin; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002412:Cations; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism*; D006461:Hemolysis / Q000187:drug effects; D007473:Ion Channels / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D012756:Sheep; D010566:Virulence Factors, Bordetella / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D002412:Cations; D017931:DNA Primers; D007473:Ion Channels; D010566:Virulence Factors, Bordetella; D018698:Glutamic Acid