Acylation of the Bordetella pertussis CyaA-hemolysin: Functional implications for efficient membrane insertion and pore formation

Kanungsuk Meetum; Chompounoot Imtong; Gerd Katzenmeier; Chanan Angsuthanasombat

doi:10.1016/j.bbamem.2016.12.011

Acylation of the Bordetella pertussis CyaA-hemolysin: Functional implications for efficient membrane insertion and pore formation

Biochim Biophys Acta Biomembr. 2017 Mar;1859(3):312-318. doi: 10.1016/j.bbamem.2016.12.011. Epub 2016 Dec 18.

Authors

Kanungsuk Meetum¹, Chompounoot Imtong², Gerd Katzenmeier¹, Chanan Angsuthanasombat³

Affiliations

¹ Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakorn Pathom 73170, Thailand.
² Division of Biology, Department of Science, Faculty of Science and Technology, Prince of Songkla University, Pattani 94000, Thailand.
³ 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.

PMID: 27993565
DOI: 10.1016/j.bbamem.2016.12.011

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 Lys⁹⁸³ and 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.

Keywords: CyaA-hemolysin; Membrane-pore formation; Palmitoylation; Planar lipid bilayers; Positively-charged patch.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Acylation
Adenylate Cyclase Toxin / chemistry
Adenylate Cyclase Toxin / genetics
Adenylate Cyclase Toxin / metabolism*
Amino Acid Sequence
Animals
Bordetella pertussis / metabolism*
Erythrocyte Membrane / chemistry
Erythrocyte Membrane / metabolism
Erythrocytes / cytology
Erythrocytes / metabolism
Hemolysis
Lipid Bilayers / chemistry
Lipid Bilayers / metabolism
Protein Binding
Recombinant Proteins / chemistry
Recombinant Proteins / isolation & purification
Recombinant Proteins / metabolism
Sequence Alignment
Sheep

Substances

Adenylate Cyclase Toxin
Lipid Bilayers
Recombinant Proteins