Effects on haemolytic activity of single proline substitutions in the Bordetella pertussis CyaA pore-forming fragment.
The recombinant Bordetella pertussis CyaA pore-forming (CyaA-PF) fragment was previously shown to be expressed separately in Escherichia coli as a soluble precursor that can be in vivo palmitoylated to exert haemolytic activity. In this study, PCR-based mutagenesis was employed to investigate the contributions to haemolysis of five predicted helices within the N-terminal hydrophobic region of the CyaA-PF fragment. Single proline substitutions were made for alanine near the centre of each predicted helix as a means of disrupting local secondary structure. All mutant proteins were over-expressed in E. coli as a 126-kDa soluble protein at levels comparable to the wild-type. Marked reductions in haemolytic activity against sheep erythrocytes of mutants, A510P, A538P, A583P and A687P pertaining to the putative helices 1(500-522), 2(529-550), 3(571-593) and 5(678-698), respectively, were observed. However, a slight decrease in haemolytic activity was found for the proline replacement in the predicted helix 4(602-627) (A616P). MALDI-TOF-MS and LC-MS-MS analyses verified the palmitoylation at Lys983 of all five mutants as identical to that of the CyaA-PF wild-type protein, indicating that toxin modification via this acylation was not affected by the mutations. Altogether, these results suggest that structural integrity of the predicted helices 1, 2, 3 and 5, but not helix 4, is important for haemolytic activity, particularly for the putative transmembrane helices 2 and 3 that might conceivably be involved in pore formation of the CyaA-PF fragment.
Powthongchin
Busaba
B
Laboratory of Molecular Biophysics and Structural Biochemistry, Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakornpathom, 73170, Thailand.
Angsuthanasombat
Chanan
C
D037361:Adenylate Cyclase Toxin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001886:Bordetella pertussis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004912:Erythrocytes / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006460:Hemolysin Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D011392:Proline / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D012756:Sheep; D014917:Whooping Cough / Q000382:microbiology*
D037361:Adenylate Cyclase Toxin; D006460:Hemolysin Proteins; D011392:Proline