Structural requirement of the hydrophobic region of the Bordetella pertussis CyaA-hemolysin for functional association with CyaC-acyltransferase in toxin acylation

Journal Article · Tue May 15 04:00:00 EDT 2018 · Biochemical and Biophysical Research Communications

DOI:https://doi.org/10.1016/J.BBRC.2018.04.007· OSTI ID:23125271

Raksanoh, Veerada ^[1]; Prangkio, Panchika ^[2]; Imtong, Chompounoot ^[3]; Thamwiriyasati, Niramon ^[4]; Suvarnapunya, Kittipong ^[5]; Shank, Lalida ^[2]; Angsuthanasombat, Chanan ^[6]

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1. Interdisciplinary Program in Biotechnology, Graduate School, Chiang Mai University, Chiang Mai 50200 (Thailand)
2. Division of Biochemistry and Biochemical Technology, Department of Chemistry, Center of Excellence in Bioresources for Agriculture, Industry and Medicine, Center of Innovation in Chemistry (PERCH-CIC), Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand)
3. Division of Biology, Department of Science, Faculty of Science and Technology, Prince of Songkla University, Pattani 94000 (Thailand)
4. Department of Medical Technology, Faculty of Allied Health Sciences, Burapha University, Chonburi 20131 (Thailand)
5. Graduate Program in Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand)
6. Bacterial Toxin Research Innovation Cluster (BRIC), Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170 (Thailand)

Highlights: • Acylation of CyaA-Hly by CyaC-acyltransferase requires AR-acylation and HP-hydrophobic regions, hence hemolysis activation. • ∼100-kDa AR-containing CyaA-RTX lacking HP cannot be acylated by functional CyaC, thus hemolytically inactive. • ∼70-kDa hemolytically inactive-CyaA-HP/BI containing both AR and HP could interact with co-expressed CyaC. • ∼20-kDa CyaA-HP comprising α1-α5 potentially interacted with CyaC via multiple H-bonding and ionic interactions. • HP is required for not only membrane-pore formation but also CyaC-association, hence toxin acylation. Previously, we demonstrated that the ∼130-kDa CyaA-hemolysin (CyaA-Hly, Met{sup 482}-Arg{sup 1706}) from Bordetella pertussis was palmitoylated at Lys{sup 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{sup 482}-Leu{sup 750}) for toxin acylation was performed. The ∼100-kDa RTX (Repeat-in-ToXin) fragment (CyaA-RTX, Ala{sup 751}-Arg{sup 1706}) containing the Lys{sup 983}-acylation region (AR, Ala{sup 751}-Gln{sup 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{sup 500}-Val{sup 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{sup 983}.

• OSTI ID: 23125271
• Journal Information: Biochemical and Biophysical Research Communications, Journal Name: Biochemical and Biophysical Research Communications Journal Issue: 4 Vol. 499; ISSN 0006-291X; ISSN BBRCA9
• Country of Publication: United States
• Language: English

Related Subjects

60 APPLIED LIFE SCIENCES
ESCHERICHIA COLI
HEMOLYSIS
HYDROGEN
MEMBRANE PORES
PROTEINS
TOXINS