Structural requirement of the hydrophobic region of the Bordetella pertussis CyaA-hemolysin for functional association with CyaC-acyltransferase in toxin acylation Veerada Raksanoh a, b, 1 , Panchika Prangkio b, 1 , Chompounoot Imtong c , Niramon Thamwiriyasati d , Kittipong Suvarnapunya e, f , Lalida Shank b, * , Chanan Angsuthanasombat f, g, ** a Interdisciplinary Program in Biotechnology, Graduate School, Chiang Mai University, Chiang Mai 50200, Thailand b 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 c Division of Biology, Department of Science, Faculty of Science and Technology, Prince of Songkla University, Pattani 94000, Thailand d Department of Medical Technology, Faculty of Allied Health Sciences, Burapha University, Chonburi 20131, Thailand e Graduate Program in Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand f Bacterial Toxin Research Innovation Cluster (BRIC), Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand g Laboratory of Molecular Biophysics and Chemical Biology, Biophysics Institute for Research and Development (BIRD), Fang, Chiang Mai 50110, Thailand article info Article history: Received 26 March 2018 Accepted 1 April 2018 Available online xxx Keywords: CyaA-RTX CyaC-acyltransferase Electrostatic potentials Hydrophobic region Palmitoylation Protein association abstract Previously, we demonstrated that the ~130-kDa CyaA-hemolysin (CyaA-Hly, Met 482 -Arg 1706 ) from Bor- detella 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 calcu- lations and molecular docking suggested that CyaA-HP comprising a1-a5 (Leu 500 -Val 698 ) can interact with CyaC through several hydrogen and ionic bonds formed between their opposite electrostatic sur- faces. 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 . © 2018 Elsevier Inc. All rights reserved. 1. Introduction Adenylate cyclase-hemolysin toxin (CyaA) is a major virulence factor secreted from Bordetella pertussis, a Gram-negative pathogen that causes whooping cough (also known as ‘pertussis’) in humans, a serious respiratory infectious disease [1]. CyaA, which belongs to the class of RTX (Repeat-in-ToXin) cytolysins, is able to facilitate respiratory tract colonization of B. pertussis by impairing defense function of host macrophages [2]. Whooping cough has now re- Abbreviations: AR, acylation region; CyaA, adenylate cyclase-hemolysin toxin; IMAC, immobilized-metal ion affinity chromatography; HP, hydrophobic region; pNPP, p-nitrophenyl palmitate; RTX, Repeat-in-ToXin. * Corresponding author. ** Corresponding author. Bacterial Toxin Research Innovation Cluster (BRIC), Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpa- thom 73170, Thailand. E-mail addresses: lalida.shank@cmu.ac.th (L. Shank), chanan.ang@mahidol.ac.th (C. Angsuthanasombat). 1 Authors with equal contributions. Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc https://doi.org/10.1016/j.bbrc.2018.04.007 0006-291X/© 2018 Elsevier Inc. All rights reserved. Biochemical and Biophysical Research Communications xxx (2018) 1e6 Please cite this article in press as: V. Raksanoh, et al., 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 (2018), https://doi.org/10.1016/j.bbrc.2018.04.007