NMR elucidation of reduced B-Z transition activity of PKZ protein kinase at high NaCl concentration Ae-Ree Lee a , Yeo-Jin Seo a , Seo-Ree Choi a , Kyoung-Seok Ryu b , Hae-Kap Cheong b , Shim Sung Lee a , Masato Katahira c , Chin-Ju Park d, * , Joon-Hwa Lee a, b, ** a Department of Chemistry and Research Institute of Natural Science, Gyeongsang National University, Gyeongnam 52828, Republic of Korea b Division of Magnetic Resonance, KBSI, Chungbuk 28119, Republic of Korea c Institute of Advanced Energy, Kyoto University, Kyoto 611-0011, Japan d Department of Chemistry, Gwangju Institute of Science and Technology, Gwangju 61005, Republic of Korea article info Article history: Received 10 November 2016 Accepted 11 November 2016 Available online 14 November 2016 Keywords: NMR Z-DNA Salt effect Z-DNA binding protein DNA-protein interaction abstract A Z-DNA binding protein (ZBP)-containing protein kinase (PKZ) in fish species has an important role in the innate immune response. Previous structural studies of the Za domain of the PKZ from Carassius auratus (caZa PKZ ) showed that the protein initially binds to B-DNA and induces B-Z transition of double stranded DNA in a salt concentration-dependent manner. However, the significantly reduced B-Z tran- sition activity of caZa PKZ at high salt concentration was not fully understood. In this study, we present the binding affinity of the protein for B-DNA and Z-DNA and characterize its extremely low B-Z transition activity at 250 mM NaCl. Our results emphasize that the B-DNA-bound form of caZa PKZ can be used as molecular ruler to measure the degree of B-Z transition. © 2016 Elsevier Inc. All rights reserved. 1. Introduction Z-DNA binding proteins (ZBPs) are critical players in various cellular functions such as RNA editing and the innate immune response [1e3]. An RNA editing enzyme (ADAR1), DNA-dependent activator of interferon-regulatory factor (DAI), the viral E3L protein, and a fish protein kinase containing a ZBP (PKZ) are examples of ZBPs which contain one or more Z-DNA binding domains [1,2,4,5]. They stabilize left-handed Z-DNA, which is a higher energy conformation than B-DNA, via complex formation [6,7]. The conserved Za domain (~60 amino acids) consists of three a helices and three b strands (a1-b1-a2-a3-b2-b3), the first half of which is considered as a helix-turn-helix fold (Fig. 1A) [8]. Several crystal structures of the Za domain in complex with dT(CG) 3 revealed that two Za molecules bind to each strand of double-stranded Z-DNA using residues in their a3 helix and their b-hairpin (b2-loop-b3) (Fig. 1B) [1,7,9,10]. The critical residues for Z-DNA binding are well conserved among various ZBPs [2]. In order to explain ZBP-DNA interaction, an active B-Z transition mechanism has been sug- gested (Fig. 1B). Firstly, the ZBP (denoted as P) binds to B-DNA (denoted as B) to form BP , and then BP is converted to ZP (Z-DNA is denoted as Z). Binding of another P to ZP generates a stable ZP 2 complex [11]. In a previous study, we applied the active B-Z transition mechanism to the function of the Za domain of the PKZ from Car- assius auratus (caZa PKZ ) [12]. The solution structure of the free form of caZa PKZ was mostly similar to its structure when bound to Z-form dT(CG) 3 , with the exception of the orientation of the b-hairpin, which is involved in a charge-charge interaction with the phos- phate backbone of the Z-DNA [12]. However, the global analysis of chemical shift perturbation data at 10 mM and 100 mM NaCl indicated that the protein had different binding affinities for B- and Z-DNA and that interaction with B-DNA was severely affected by the concentration of NaCl ([NaCl]). By monitoring ellipticity at 255 nm for the B-Z transition with time course CD spectroscopy at various [NaCl], it has been shown that the B-Z transition activity of caZa PKZ is strongly salt concentration-dependent, unlike other ZBPs, and that high salt concentrations (tested up to 250 mM NaCl) produce extremely low activity [10]. In order to investigate the B-Z transition induced by caZa PKZ at high [NaCl], we analyzed the imino proton spectra of dT(CG) 3 Abbreviations: caZa PKZ , the Za domain of Carassius auratus protein kinase Z; ZBP, Z-DNA binding protein. * Corresponding author. ** Corresponding author. Department of Chemistry and RINS, Gyeongsang Na- tional University, Gyeongnam 52828, Republic of Korea. E-mail addresses: cjpark@gist.ac.kr (C.-J. Park), joonhwa@gnu.ac.kr (J.-H. Lee). Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc http://dx.doi.org/10.1016/j.bbrc.2016.11.064 0006-291X/© 2016 Elsevier Inc. All rights reserved. Biochemical and Biophysical Research Communications 482 (2017) 335e340