Molecules and Cells Insight into Structural Aspects of Histidine 284 of Daphnia magna Arginine Kinase Zhili Rao1,4, So Young Kim1,4, Xiaotong Li1, Da Som Kim1, Yong Ju Kim2,3,*, and Jung Hee Park1,3,* 1Division of Biotechnology, College of Environmental & Bioresources Sciences, Jeonbuk National University, Iksan 54596, Korea, 2Department of Herbal Medicine Resources, College of Environmental and Bioresource Sciences, Jeonbuk National University, Iksan 54596, Korea, 3Advanced Institute of Environment and Bioscience, College of Environmental & Bioresources Sciences, Jeonbuk National University, Iksan 54596, Korea, 4These authors contributed equally to this work. *Correspondence: [email protected] (YJK); [email protected] (JHP) https://doi.org/10.14348/molcells.2020.0136 www.molcells.org Arginine kinase (AK), a bioenergy-related enzyme, is INTRODUCTION distributed widely in invertebrates. The role of highly conserved histidines in AKs is still unascertained. In this study, Phosphagen kinases (PKs) are a family of phosphotransfer- the highly conserved histidine 284 (H284) in AK of Daphnia ases that reversibly catalyze the delivery of high-energy phos- magna (DmAK) was replaced with alanine to elucidate the phoryl groups between ATP and guanidine derivatives. The role of H284. We examined the alteration of catalytic activity phosphorylated guanidines, also known as phosphagens, are and structural changes of H284A in DmAK. The catalytic a type of high-energy compounds that play an important role activity of H284A was reduced dramatically compared to in cellular energy homeostasis in both the muscles and the that in wild type (WT). Thus the crystal structure of H284A brain by providing a way to store high-energy phosphates displayed several structural changes, including the alteration as a metastable compound (Ellington, 2001). PKs are wide- of D324, a hydrogen-bonding network around H284, and spread in all types of animal cells that require considerable the disruption of π-stacking between the imidazole group of amounts of energy. Eight PKs have been reported in inverte- the H284 residue and the adenine ring of ATP. These findings brates (Ellington, 1989; Uda et al., 2005). One of these, ar- suggest that such alterations might affect a conformational ginine kinase (AK), is distributed widely in invertebrates, and change of the specific loop consisting of G310-V322 at its activity has also been observed in arthropods, mollusks, the antiparallel β-sheet region. Thus, we speculated that nematodes, protozoans, and some bacteria (Ellington, 1989; the H284 residue might play an important role in the Newsholme et al., 1978). conformational change of the specific loop when ATP binds Arginine, which is an essential amino acid in many or- to the substrate-binding site of DmAK. ganisms, is produced as an intermediate in two regulatory cycles: the urea and the nitric oxide cycles (Noh et al., 2002). Keywords: arginine kinase, crystallization, kinetics, point This intermediate is also used by AK to maintain energy mutation, X-ray crystallography homeostasis in cells. AK requires both arginine and ATP as bi-substrates and plays a key role in catalyzing the reversible transfer of the γ-phosphoryl group of ATP to the guanidine functional group of arginine (Adeyemi and Whiteley, 2014; Alonso et al., 2001; Hansen and Knowles, 1981; Wyss and Received 19 June, 2020; revised 29 July, 2020; accepted 10 August, 2020; published online 31 August, 2020 eISSN: 0219-1032 ©The Korean Society for Molecular and Cellular Biology. All rights reserved. cc This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/. 784 Mol. Cells 2020; 43(9): 784-792 The Role of H284 in Structural Change of the Specific Loop Zhili Rao et al. Kaddurah-Daouk, 2000; Zhou et al., 1998). In particular, tion of H284A to DmAK, some structural alterations of the AK activity is required for muscle contraction and the gen- surrounding residue were detected, i.e., the disruption of eration of electrical conductance in nerves. Many functional π-stacking between the imidazole group of the H284 residue and structural studies of AKs from diverse organisms have and the adenine ring of ATP, the alteration of D324, and a been reported in a substrate-binding-dependent manner hydrogen-bonding network around H284. These findings (Azzi et al., 2004; Brown and Grossman, 2004; Fernandez may be important clues for explaining the effects of H284A et al., 2007; Niu et al., 2011; Pruett et al., 2003; Watts et on the reduction of activity compared with WT. Therefore, al., 1980). The overall topology of AKs commonly has two our results provide a clue to improve our understanding of different domains: an N-terminal domain, which consists of the bioenergetics mechanism. small α-helices, and a C-terminal domain, which comprises an eight-strand antiparallel β-sheet connected by α-helices. In MATERIALS AND METHODS AKs, the N-terminal domain can interact with arginine (i.e., either L-arginine or phosphoarginine), whereas the central Cloning, expression, and purification of DmAK WT and role of the C-terminal domain is nucleotide-binding (i.e., ei- H284A ther ATP or ADP) (Fernandez et al., 2007; Zhou et al., 1998). The DmAK gene was synthesized according to the full-length In the structural comparison between Trypanosoma cruzi open reading frame (GenBank accession No. AID69955.1) AKapo (Tc AKapo) and Limulus Polyphemus AKholo (LpAKholo), from National Center for Biotechnology Information (NCBI; some structural changes accompanied by rigid-body motion http://www.ncbi.nlm.nih.gov). The synthesized gene was of the helix and the antiparallel β-sheet outward from the nu- amplified by a standard PCR method with a primer set (5′- cleotide-binding site in the C-terminal domain were observed GCACTCCATATGCATCACCATCATCATCATGTGGAC-3′ and 5′- significantly (Clark et al., 2012; Fernandez et al., 2007). GCACTCGCGG CCGCTTATGCGGC TTC-3′) and inserted into In addition, the structural changes in the substrate-bound an expression vector (pET30a; New England Biolabs, USA) (holo) and the substrate-free (apo) state of AK have been using NdeI and NotI restriction enzyme sites. A six-His tag was confirmed by joint crystallographic and NMR residual dipolar fused to the N-terminus of the gene to facilitate protein pu- coupling analysis (Niu et al., 2011). Thus, the results indicated rification. The mutation was introduced by modified overlap that the two different AK conformations might derive from PCR using a single site mismatched primer against H284 (5′- substrate-induced motion. GCAGTGCAATGGCCACTGAGGCCC-3′). The recombinant Several highly conserved residues in the arginine and ATP construct was transformed into Escherichia coli BL21 (DE3) binding sites that alter AK enzyme activity have been report- cells. Then, overnight cultures of the transformed cells were ed (Guo et al., 2004; Strong and Ellington, 1996; Takeuchi et transferred to 1 L of LB broth (BD Bioscience, USA) contain- al., 2004; Wu et al., 2014). In Nautilus and Stichophus AKs, ing kanamycin (Duchefa Biochemie, The Netherlands), with the mutations of D62G and R193G accompanying the disap- a final concentration of 50 μg/ml, and incubated at 37°C pearance of the salt bridge showed considerably decreased and 200 rpm until reaching the optical density of 0.6 at 600 activity. Hence, the mutagenesis of both D62G and R193G nm (OD600). Large quantities of recombinant DmAK proteins has been proposed to be associated with structural stability were obtained by inducing the expression of DmAK WT and and bi-substrates’ affinity (Suzuki et al., 2000a; 2000b). On H284A with 0.5 mM isopropyl β-D-1-thiogalactopyranoside the other hand, the mutations of C271, P272 and T273 (IPTG) (Duchefa Biochemie) for 4 h at 18°C and 170 rpm. replaced with alanine, which are located at the hinge loop The harvested cells were resuspended in 20 ml of buffer (10 in AKs, showed reduced activity in the previous studies (Liu mM Tris-HCl, pH 8.0, 100 mM NaCl) and lysed for 2 min by et al., 2011; Strong and Ellington, 1996; Wu et al., 2008; sonication (4 s sonication and 4 s rest) (Sonics and Materi- 2014). These findings revealed an alteration of enzyme activi- als, USA). The cellular debris was removed by centrifugation ty due to structural changes at the arginine binding site. Thus, (12,000 rpm, 4°C) for 20 min, and then the supernatant was these residues were confirmed as key players in the substrate purified using affinity chromatography with a HisTrap FF col- synergism between the arginine residues (L-arginine and umn (GE Healthcare, USA) (Rao et al., 2019). To ensure the phosphoarginine) of AK and their constraining position (Wu high purity of DmAK WT and H284A, the HiTrap Q FF column et al., 2014). In the transition state analog (TSA) including (GE Healthcare) was used with a double gradient elution nitrate (NO3), the crystal structure of AK from horseshoe crab buffer system (10 mM Tris-HCl, pH 7.0, 1 M NaCl). The puri- revealed the relationship between nitrate and the bi-sub- fied DmAK was analyzed by 12% SDS-PAGE (Supplementary strate (Zhou et al., 1998). The substrate arginine showed Method), and Bradford’s method was employed to ascertain two salt bridges with E225 and E314. Hence, the variants of the protein concentration (Bradford, 1976). E225 and E314 exhibited alteration of catalytic activity with a reduced rate of production of phosphoarginine and ADP Enzyme assay and determination of kinetic parameters (Pruett et al., 2003). To ascertain the optimal pH and temperature, AK activity was Although several mutagenesis studies have reported highly conducted as described previously (Li et al., 2006; Pereira et conserved residues at the arginine binding site of AKs, a role al., 2000; Wu et al., 2014).