title: Functional significance of the highly conserved Glu(570) in the putative pore-forming helix 3 of the Bordetella pertussis haemolysin toxin.
abstract: Adenylate cyclase-haemolysin toxin (CyaA) is a virulence factor secreted from the etiologic agent of whooping cough, Bordetella pertussis. Previously, the haemolysin or pore-forming domain (CyaA-PF) has been shown to cause cell lysis of sheep erythrocytes independently, and the predicted helix 3((570-593)) within the PF-hydrophobic stretch could be a pore-lining constituent. Here, a plausible involvement in haemolytic activity of polar or charged residues (Glu(570), Gln(574), Glu(581), Ser(584) and Ser(585)) lining the hydrophilic side of CyaA-PF helix 3 was investigated via single-alanine substitutions. All the 126-kDa mutant proteins over-expressed in Escherichia coli were verified for toxin acylation as the results are corresponding to the wild-type toxin. When haemolytic activity of E. coli lysates containing soluble mutant proteins was tested against sheep erythrocytes, the importance of Glu(570), which is highly conserved among the pore-forming RTX cytotoxin family, was revealed for pore formation, conceivably for a general pore-lining residue involved in ion conduction.
authors.affiliation: Laboratory of Molecular Biophysics and Structural Biochemistry, Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand.
authors.forename: Chattip
authors.initials: C
authors.lastname: Kurehong
authors.forename: Busaba
authors.initials: B
authors.lastname: Powthongchin
authors.forename: Niramon
authors.initials: N
authors.lastname: Thamwiriyasati
authors.forename: Chanan
authors.initials: C
authors.lastname: Angsuthanasombat
mesh_terms: D000215:Acylation; D037361:Adenylate Cyclase Toxin / Q000737:chemistry / Q000235:genetics* / Q000633:toxicity*; D000818:Animals; D001886:Bordetella pertussis / Q000201:enzymology*; D017124:Conserved Sequence / Q000235:genetics; D004912:Erythrocytes / Q000187:drug effects; D004926:Escherichia coli; D018698:Glutamic Acid / Q000737:chemistry*; D006460:Hemolysin Proteins / Q000737:chemistry / Q000235:genetics*; D016296:Mutagenesis; D017433:Protein Structure, Secondary / Q000235:genetics*; D012756:Sheep
chemical_list: D037361:Adenylate Cyclase Toxin; D006460:Hemolysin Proteins; D018698:Glutamic Acid