title: Acylation of the Bordetella pertussis CyaA-hemolysin: Functional implications for efficient membrane insertion and pore formation.
abstract: Previously, the ~130-kDa CyaA-hemolysin domain (CyaA-Hly) from Bordetella pertussis co-expressed with CyaC-acyltransferase in Escherichia coli was demonstrated to be palmitoylated at Lys983and thus activated its hemolytic activity against target erythrocytes. Here, we report the functional importance of Lys983-palmitoylation for membrane insertion and pore formation of CyaA-Hly. Intrinsic fluorescence emissions of both non-acylated CyaA-Hly (NA/CyaA-Hly) and CyaA-Hly were indistinguishable, suggesting no severe conformational change upon acylation at Lys983. Following pre-incubation of sheep erythrocytes with NA/CyaA-Hly, there was a drastic decrease in CyaA-Hly-induced hemolysis. Direct interactions between NA/CyaA-Hly and target erythrocyte membranes were validated via membrane-binding assays along with Western blotting, suggestive of acylation-independent capability of NA/CyaA-Hly to interact with erythrocyte membranes. As compared with CyaA-Hly, NA/CyaA-Hly displayed a slower rate of incorporation into DOPC:DOPE:Ch or DiPhyPC bilayers under symmetrical conditions (1M KCl, 10mM HEPES, pH7.4) and formed channels exhibiting different conductance. Further analysis revealed that channel-open lifetime in DOPC:DOPE:Ch bilayers of NA/CyaA-Hly was much shorter than that of the acylated form, albeit slightly shorter lifetime found in DiPhyPC bilayers. Sequence alignments of the Lys983-containing CyaA-segment with those of related RTX-cytolysins revealed a number of highly conserved hydrophobic residues and a Lys/Arg cluster that is predicted be important for toxin-membrane interactions. Altogether, our data disclosed that the Lys983-linked palmitoyl group is not directly involved in either binding to target erythrocyte membranes or toxin-induced channel conductivity, but rather required for efficient membrane insertion and pore formation of the acylated CyaA-Hly domain.
authors.affiliation: Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakorn Pathom 73170, Thailand.
authors.forename: Kanungsuk
authors.initials: K
authors.lastname: Meetum
authors.affiliation: Division of Biology, Department of Science, Faculty of Science and Technology, Prince of Songkla University, Pattani 94000, Thailand.
authors.forename: Chompounoot
authors.initials: C
authors.lastname: Imtong
authors.affiliation: Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakorn Pathom 73170, Thailand.
authors.forename: Gerd
authors.initials: G
authors.lastname: Katzenmeier
authors.affiliation: Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakorn Pathom 73170, Thailand; Laboratory of Molecular Biophysics and Structural Biochemistry, Biophysics Institute for Research and Development (BIRD), Bangkok 10160, Thailand. Electronic address: chanan.ang@mahidol.ac.th.
authors.forename: Chanan
authors.initials: C
authors.lastname: Angsuthanasombat
mesh_terms: D000215:Acylation; D037361:Adenylate Cyclase Toxin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001886:Bordetella pertussis / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000737:chemistry / Q000378:metabolism; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism; D006461:Hemolysis; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D012756:Sheep
chemical_list: D037361:Adenylate Cyclase Toxin; D008051:Lipid Bilayers; D011994:Recombinant Proteins
keywords: CyaA-hemolysin; Membrane-pore formation; Palmitoylation; Planar lipid bilayers; Positively-charged patch