Publication: Structural requirement of the hydrophobic region of the Bordetella pertussis CyaA-hemolysin for functional association with CyaC-acyltransferase in toxin acylation
2
Issued Date
2018-05-23
Resource Type
ISSN
10902104
0006291X
0006291X
Other identifier(s)
2-s2.0-85044991866
Rights
Mahidol University
Rights Holder(s)
SCOPUS
Bibliographic Citation
Biochemical and Biophysical Research Communications. Vol.499, No.4 (2018), 862-867
Suggested Citation
Veerada Raksanoh, Panchika Prangkio, Chompounoot Imtong, Niramon Thamwiriyasati, Kittipong Suvarnapunya, Lalida Shank, Chanan Angsuthanasombat Structural requirement of the hydrophobic region of the Bordetella pertussis CyaA-hemolysin for functional association with CyaC-acyltransferase in toxin acylation. Biochemical and Biophysical Research Communications. Vol.499, No.4 (2018), 862-867. doi:10.1016/j.bbrc.2018.04.007 Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/45161
Research Projects
Organizational Units
Authors
Journal Issue
Thesis
Title
Structural requirement of the hydrophobic region of the Bordetella pertussis CyaA-hemolysin for functional association with CyaC-acyltransferase in toxin acylation
Abstract
© 2018 Elsevier Inc. Previously, we demonstrated that the ∼130-kDa CyaA-hemolysin (CyaA-Hly, Met 482 -Arg 1706 ) from Bordetella pertussis was palmitoylated at Lys 983 when co-expressed with CyaC-acyltransferase in Escherichia coli, and thus activated its hemolytic activity. Here, further investigation on a possible requirement of the N-terminal hydrophobic region (HP, Met 482 -Leu 750 ) for toxin acylation was performed. The ∼100-kDa RTX (Repeat-in-ToXin) fragment (CyaA-RTX, Ala 751 -Arg 1706 ) containing the Lys 983 -acylation region (AR, Ala 751 -Gln 1000 ), but lacking HP, was co-produced with CyaC in E. coli. Hemolysis assay indicated that CyaA-RTX showed no hemolytic activity. Additionally, MALDI-TOF/MS and LC-MS/MS analyses confirmed that CyaA-RTX was non-acylated, although the co-expressed CyaC-acyltransferase was able to hydrolyze its chromogenic substrate−p-nitrophenyl palmitate and acylate CyaA-Hly to become hemolytically active. Unlike CyaA-RTX, the ∼70-kDa His-tagged CyaA-HP/BI fragment which is hemolytically inactive and contains both HP and AR was constantly co-eluted with CyaC during IMAC-purification as the presence of CyaC was verified by Western blotting. Such potential interactions between the two proteins were also revealed by semi-native PAGE. Moreover, structural analysis via electrostatic potential calculations and molecular docking suggested that CyaA-HP comprising α1-α5 (Leu 500 -Val 698 ) can interact with CyaC through several hydrogen and ionic bonds formed between their opposite electrostatic surfaces. Overall, our results demonstrated that the HP region of CyaA-Hly is conceivably required for not only membrane-pore formation but also functional association with CyaC-acyltransferase, and hence effective palmitoylation at Lys 983 .