Contents lists available at ScienceDirect Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi Role of the glutaredoxin domain and FAD in the stabilization of thioredoxin glutathione reductase Parismita Kalita a , Harish Shukla a , Kundlik Gadhave b , Rajanish Giri b , Timir Tripathi a,* a Molecular and Structural Biophysics Laboratory, Department of Biochemistry, North-Eastern Hill University, Shillong- 793022, India b School of Basic Sciences, Indian Institute of Technology Mandi, Kamand, Himachal Pradesh, 175005, India ARTICLE INFO Keywords: Parasite Liver fluke Thioredoxin glutathione reductase Glutathione reductase Thioredoxin reductase Activity Stability FAD Fluorescence ABSTRACT Thioredoxin glutathione reductase (TGRsec) is a multi-domain flavoprotein that plays a principal role in redox homeostasis maintenance. We have previously demonstrated the role of selenocysteine in maintaining TGRsec structure-function, but the role of the glutaredoxin (Grx) domain and FAD is still unclear. In the present study, the urea-induced unfolding of recombinant Fasciola gigantica TGRsec (FgTGRsec) and its N-terminal truncated variant (ΔNTD-FgTGRsec) were examined to understand the role of the Grx domain and FAD in the stabilization of FgTGRsec and ΔNTD-FgTGRsec. Our results showed that both proteins underwent unfolding in a three state manner. First, the protein undergoes a conformational transition rendering a near-native state with no FAD bound, and then full unfolding of the apo-dimer occurs without dissociation. The Grx domain stabilized the global FgTGRsec structure and positively regulated FgTGRsec activity, and alteration in the FAD micro- environment was directly proportional to the loss of thioredoxin reductase (TrxR) and glutathione reductase activities. Based on these results, we concluded that the Grx domain stabilizes the full-length FgTGRsec protein for efficient catalysis. Thus, we suggest that in platyhelminth parasites, during evolution, the Grx domain merged with the TrxR domain to confer higher catalytic activity and provide additional structural stability to the full- length TGR. 1. Introduction In vivo, proteins are mostly present in a specific “functionally active” 3D conformation with low energy and entropy, called the native or folded state, which is primarily determined by its amino acid sequence and the surrounding environment. A structurally unfolded and/or de- natured protein with a high-energy conformation may result from various solvents, detergents, and chemicals, such as sodium dodecyl sulfate, alcohol, urea, and guanidine hydrochloride (GdnHCl), that can disturb the protein structure and stability [1,2]. The denaturants can either directly interact with the chemical groups of the protein, thereby enabling covalent modification or perturbing the electrostatic interac- tions [3,4], or cause alteration in the solvent it is kept [5] by forming non-covalent cross-links with the protein [6]. The precise mechanism of protein denaturation/unfolding by chemical denaturants is still a de- bate, but urea-mediated denaturation of proteins is known to proceed via two mechanisms: (i) by solvation of hydrophobic residues due to the disruption of water structure or (ii) by formation of hydrogen bonds and Vander Waals interaction with the protein side chains and back- bone [7–9]. Fascioliasis, caused by the genus Fasciola, is a neglected food-borne trematodiasis, which causes both livestock and economic losses in the affected countries. Globally, fascioliasis affects over 600 million ani- mals, causing an economic loss of > US$3 billion p.a. in livestock production [10,11]. In Africa, the Middle East, and Southeast Asia, approximately 25%–100% of the cattle acquire fascioliasis annually, in addition to approximately 70% humans in temperate countries, who acquire fascioliasis mainly through the consumption of raw or under- cooked fish, meat, or leafy vegetables [11]. To date, approximately 2.4–17 million people have been infected globally, with approximately 180 million people at risk of infection [10]. Human fascioliasis is highly prevalent in the Bolivian Altiplano, and many cases have been reported from Africa, Europe, the Middle East (including Egypt), Southeast Asia, and Latin America [12,13]. The recommended treatment for fascioliasis is the administration of triclabendazole (TCZ; 10 mg/kg), which has been available since several decades [14]. In 2016, approximately 600,000 individuals worldwide reportedly received TCZ treatment. In addition, there are reports on increased TCZ resistance in these para- sites, suggesting the urgent need for developing new therapeutics against fascioliasis [15–18]. https://doi.org/10.1016/j.abb.2018.09.002 Received 27 June 2018; Received in revised form 4 September 2018; Accepted 7 September 2018 * Corresponding author. E-mail addresses: timir.tripathi@gmail.com, ttripathi@nehu.ac.in (T. Tripathi). Archives of Biochemistry and Biophysics 656 (2018) 38–45 Available online 08 September 2018 0003-9861/ © 2018 Elsevier Inc. All rights reserved. T