Contents lists available at ScienceDirect Toxicon journal homepage: www.elsevier.com/locate/toxicon Synthesis, folding, structure and activity of a predicted peptide from the sea anemone Oulactis sp. with an ShKT fold Bankala Krishnarjuna a,1 , Jessica Villegas-Moreno a,b,1 , Michela L. Mitchell a , Agota Csoti c,d , Steve Peigneur e , Carlos Amero b , Michael W. Pennington f , Jan Tytgat e , Gyorgy Panyi c,d , Raymond S. Norton a,* a Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia b Centro de Investigaciones Químicas, Universidad Autónoma del Estado de Morelos, Cuernavaca, Mexico c Department of Biophysics and Cell Biology, University of Debrecen, 4032 Debrecen, Hungary d MTA-DE-NAP B Ion Channel Structure-Function Research Group, RCMM, University of Debrecen, 4032 Debrecen, Hungary e Toxicology and Pharmacology, University of Leuven, O&N 2, 3000, Leuven, Belgium f Peptides International, Louisville, KY 40299, USA ARTICLE INFO Keywords: OspTx2b Cysteine-rich peptide Sea anemone Structure NMR spectroscopy Potassium channel ABSTRACT Sea anemone venom is rich in bioactive compounds, including peptides containing multiple disulfide bridges. In a transcriptomic study on Oulactis sp., we identified the putative 36-residue peptide, OspTx2b, which is an isoform of the K V channel blocker OspTx2a (Sunanda P et al. [2018] Identification, chemical synthesis, structure and function of a new K V 1 channel blocking peptide from Oulactis sp. Peptide Science, in press). As OspTx2b contains a ShK/BgK-like cysteine framework, with high amino acid sequence similarity to BgK, we were in- terested to investigate its structure and function. The solution structure of OspTx2b was determined using nu- clear magnetic resonance spectroscopy. OspTx2b does indeed possess a BgK-like scaffold, with the same disulfide bond connectivities. The orientation of the Lys-Tyr dyad in OspTx2b is more similar to that in ShK than in BgK. However, it failed to show against a range of voltage-gated potassium channels in Xenopus oocytes and human T lymphocytes. OspTx2b also showed no growth inhibitory activity against several strains of bacteria and fungi. Having a BgK-like fold with the Lys-Tyr dyad but no BgK-like activity highlights the importance of key amino acid residues in BgK that are missing in OspTx2b. The lack of activity against the K V channels assessed in this study emphasises that the ShK/BgK scaffold is capable of supporting functional activity beyond potassium channel blockade. 1. Introduction Sea anemone venom is a rich source of bioactive compounds, in- cluding peptide toxins containing multiple disulfide bonds (Frazão et al., 2012; Norton, 2009; Prentis et al., 2018; Shiomi, 2009). Many of the known peptide toxins are selective and potent inhibitors or mod- ulators of various ion channels (Norton and Chandy, 2017). The sea anemone peptide ShK (from Stichodactyla helianthus)(Castañeda et al., 1995) is a potent blocker of the voltage-gated potassium channel K V 1.3 (Kalman et al., 1998). As this channel regulates the activation and proliferation of human effector memory T lymphocytes (T EM ), which play an important role in causing inflammation in autoimmune diseases (Beeton et al., 2011; Chi et al., 2012; Feske et al., 2015), blocking K V 1.3 channels represents a promising approach for the treatment of these conditions (Beeton et al., 2011; Chi et al., 2012; Panyi et al., 2006). An analogue of ShK is currently under clinical development for the treat- ment of autoimmune diseases such as psoriasis (Pennington et al., 2009; Tarcha et al., 2017). Peptide toxins from different venomous animals may show amino acid sequence and structural homology, yet can have divergent biolo- gical functions. Conversely, peptides with limited amino acid sequence and structural homology may converge on a common function. For example, the sea anemone peptide toxins ShK and BgK (from Bunodosoma granulifera) have completely different structures from the scorpion peptide HsTX1 (from Heterometrus spinnifer)(Lebrun et al., 1997), but all three are potent blockers of K V 1.3 channels, as they share a functional Lys-Tyr dyad (Dauplais et al., 1997; Mouhat et al., 2005; Norton and Chandy, 2017). However, AsK132958[Q20Y], a mutant of https://doi.org/10.1016/j.toxicon.2018.05.006 Received 15 March 2018; Received in revised form 8 May 2018; Accepted 13 May 2018 * Corresponding author. Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia. 1 These authors contributed equally to this work. E-mail address: ray.norton@monash.edu (R.S. Norton). Toxicon 150 (2018) 50–59 0041-0101/ © 2018 Published by Elsevier Ltd. T