title: Segments crucial for membrane translocation and pore-forming activity of Bordetella adenylate cyclase toxin.
abstract: Bordetella adenylate cyclase toxin-hemolysin (CyaA, AC-Hly, or ACT) permeabilizes cell membranes by forming small cation-selective (hemolytic) pores and subverts cellular signaling by delivering into host cells an adenylate cyclase (AC) enzyme that converts ATP to cAMP. Both AC delivery and pore formation were previously shown to involve a predicted amphipathic alpha-helix(502-522) containing a pair of negatively charged Glu(509) and Glu(516) residues. Another predicted transmembrane alpha-helix(565-591) comprises a Glu(570) and Glu(581) pair. We examined the roles of these glutamates in the activity of CyaA. Substitutions of Glu(516) increased specific hemolytic activity of CyaA by two different molecular mechanisms. Replacement of Glu(516) by positively charged lysine residue (E516K) increased the propensity of CyaA to form pores, whereas proline (E516P) or glutamine (E516Q) substitutions extended the lifetime of open single pore units. All three substitutions also caused a drop of pore selectivity for cations. Substitutions of Glu(570) and Glu(581) by helix-breaking proline or positively charged lysine residue reduced (E570K, E581P) or ablated (E570P, E581K) AC membrane translocation. Moreover, E570P, E570K, and E581P substitutions down-modulated also the specific hemolytic activity of CyaA. In contrast, the E581K substitution enhanced the hemolytic activity of CyaA 4 times, increasing both the frequency of formation and lifetime of toxin pores. Negative charge at position 570, but not at position 581, was found to be essential for cation selectivity of the pore, suggesting a role of Glu(570) in ion filtering inside or close to pore mouth. The pairs of glutamate residues in the predicted transmembrane segments of CyaA thus appear to play a key functional role in membrane translocation and pore-forming activities of CyaA.
authors.affiliation: Institute of Microbiology of the Academy of Sciences of the Czech Republic, Videnska 1083, CZ-142 20 Prague 4, Czech Republic.
authors.forename: Marek
authors.initials: M
authors.lastname: Basler
authors.forename: Oliver
authors.initials: O
authors.lastname: Knapp
authors.forename: Jiri
authors.initials: J
authors.lastname: Masin
authors.forename: Radovan
authors.initials: R
authors.lastname: Fiser
authors.forename: Elke
authors.initials: E
authors.lastname: Maier
authors.forename: Roland
authors.initials: R
authors.lastname: Benz
authors.forename: Peter
authors.initials: P
authors.lastname: Sebo
authors.forename: Radim
authors.initials: R
authors.lastname: Osicka
mesh_terms: D037361:Adenylate Cyclase Toxin / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D019943:Amino Acid Substitution; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D001884:Bordetella / Q000201:enzymology / Q000235:genetics; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006461:Hemolysis / Q000187:drug effects / Q000235:genetics; D020125:Mutation, Missense; D021381:Protein Transport / Q000235:genetics; D012756:Sheep; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics
chemical_list: D037361:Adenylate Cyclase Toxin; D001426:Bacterial Proteins