title: Functional importance of the Gly cluster in transmembrane helix 2 of the Bordetella pertussis CyaA-hemolysin: Implications for toxin oligomerization and pore formation.
abstract: Adenylate cyclase-hemolysin (CyaA) is a major virulence factor of Bordetella pertussis causing whooping cough in humans. We previously showed that two transmembrane helices (α2 and α3) in the hemolysin domain (CyaA-Hly) are crucially involved in hemolytic activity. Here, PCR-based substitutions were employed to investigate a potential involvement in hemolysis of a series of four Gly residues (Gly(530), Gly(533), Gly(537) and Gly(544)) which map onto one face of a helical wheel plot of pore-lining helix 2. All CyaA-Hly mutant toxins were over-expressed in Escherichia coli as 126-kDa soluble proteins at levels comparable to the wild-type toxin. A drastic reduction in hemolytic activity against sheep erythrocytes was observed for three CyaA-Hly mutants, i.e. G530A, G533A and G537A, but not G544A, suggesting a functional importance of the Gly(530)_Gly(533)_Gly(537) cluster. A homology-based structure of the α2-loop-α3 hairpin revealed that this crucial Gly cluster arranged as a GXXGXXXG motif is conceivably involved in helix-helix association. Furthermore, a plausible pore model comprising three α2-loop-α3 hairpins implicated that Gly(530)XXGly(533)XXXGly(537) could function as an important framework for toxin oligomerization. Altogether, our present data signify for the first time that the Gly(530)_Gly(533)_Gly(537) cluster in transmembrane helix 2 serves as a crucial constituent of the CyaA-Hly trimeric pore structure.
authors.affiliation: Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom 73170, Thailand.
authors.forename: Sirikran
authors.initials: S
authors.lastname: Juntapremjit
authors.affiliation: Department of Medical Technology, Faculty of Allied Health Sciences, Burapha University, Chonburi 20131, Thailand.
authors.forename: Niramon
authors.initials: N
authors.lastname: Thamwiriyasati
authors.affiliation: Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom 73170, Thailand.
authors.forename: Chattip
authors.initials: C
authors.lastname: Kurehong
authors.affiliation: Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand.
authors.forename: Panchika
authors.initials: P
authors.lastname: Prangkio
authors.affiliation: Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand.
authors.forename: Lalida
authors.initials: L
authors.lastname: Shank
authors.affiliation: Department of Biopharmacy, Faculty of Pharmacy, Silpakorn University, Nakhon Pathom 73000, Thailand. Electronic address: powthongchin_b@su.ac.th.
authors.forename: Busaba
authors.initials: B
authors.lastname: Powthongchin
authors.affiliation: Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Nakhon 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: D037361:Adenylate Cyclase Toxin / Q000737:chemistry* / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001886:Bordetella pertussis / Q000737:chemistry*; D004912:Erythrocytes / Q000187:drug effects; D005998:Glycine / Q000737:chemistry / Q000502:physiology*; D006461:Hemolysis / Q000187:drug effects; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D000072417:Protein Domains; D020539:Sequence Analysis, Protein; D012756:Sheep
chemical_list: D037361:Adenylate Cyclase Toxin; D005998:Glycine
keywords: Adenylate cyclase-hemolysin; GXXGXXXG motif; Helix–helix association; Hemolytic activity; Trimeric pore model