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Functional importance of the Gly cluster in transmembrane helix 2 of the Bordetella pertussis CyaA-hemolysin: Implications for toxin oligomerization and pore formation

Author:
Juntapremjit, Sirikran, Thamwiriyasati, Niramon, Kurehong, Chattip, Prangkio, Panchika, Shank, Lalida, Powthongchin, Busaba, Angsuthanasombat, Chanan
Source:
Toxicon 2015 v.106 pp. 14-19
ISSN:
0041-0101
Subject:
Bordetella pertussis, Escherichia coli, erythrocytes, gene overexpression, hemolysins, hemolysis, humans, models, mutants, oligomerization, polymerase chain reaction, proteins, sheep, toxins, virulence, whooping cough
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 (Gly530, Gly533, Gly537 and Gly544) 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 Gly530_Gly533_Gly537 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 Gly530XXGly533XXXGly537 could function as an important framework for toxin oligomerization. Altogether, our present data signify for the first time that the Gly530_Gly533_Gly537 cluster in transmembrane helix 2 serves as a crucial constituent of the CyaA-Hly trimeric pore structure.
Agid:
5451264