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Please use this identifier to cite or link to this item: http://repository.li.mahidol.ac.th/dspace/handle/123456789/45161
Title: Structural requirement of the hydrophobic region of the Bordetella pertussis CyaA-hemolysin for functional association with CyaC-acyltransferase in toxin acylation
Authors: Veerada Raksanoh
Panchika Prangkio
Chompounoot Imtong
Niramon Thamwiriyasati
Kittipong Suvarnapunya
Lalida Shank
Chanan Angsuthanasombat
Mahidol University
Faculty of Medicine, Siriraj Hospital, Mahidol University
Burapha University
Prince of Songkla University
Chiang Mai University
Biophysics Institute for Research and Development (BIRD)
Keywords: Biochemistry, Genetics and Molecular Biology
Issue Date: 23-May-2018
Citation: Biochemical and Biophysical Research Communications. Vol.499, No.4 (2018), 862-867
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 .
URI: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85044991866&origin=inward
http://repository.li.mahidol.ac.th/dspace/handle/123456789/45161
ISSN: 10902104
0006291X
Appears in Collections:Scopus 2018

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