1 Scientific RepoRts | 7:45466 | DOI: 10.1038/srep45466 www.nature.com/scientificreports structural mechanisms for α-conotoxin activity at the human α3β4 nicotinic acetylcholine receptor Nikita Abraham, Michael Healy, Lotten Ragnarsson, Andreas Brust, paul F. Alewood & Richard J. Lewis Nicotinic acetylcholine receptors (nAChR) are therapeutic targets for a range of human diseases. α-Conotoxins are naturally occurring peptide antagonists of nAChRs that have been used as pharmacological probes and investigated as drug leads for nAChR related disorders. However, α-conotoxin interactions have been mostly characterised at the α7 and α3β2 nAChRs, with interactions at other subtypes poorly understood. this study provides novel structural insights into the molecular basis for α-conotoxin activity at α3β4 nAChR, a therapeutic target where subtype specifc antagonists have potential to treat nicotine addiction and lung cancer. A co-crystal structure of α-conotoxin LsIA with Lymnaea stagnalis acetylcholine binding protein guided the design and functional characterisations of LsIA analogues that identifed the minimum pharmacophore regulating α3β4 antagonism. Interactions of the LsIA R10F with β4 K57 and the conserved –NN– α-conotoxin motif with β4 I77 and I109 conferred α3β4 activity to the otherwise inactive LsIA. Using these structural insights, we designed LsIA analogues with α3β4 activity. This new understanding of the structural basis of protein-protein interactions between α-conotoxins and α3β4 may help rationally guide the development of α3β4 selective antagonists with therapeutic potential. Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand gated ion channels involved in the modulation of neurotransmission in the central and peripheral nervous system 1–4 . Te nAChR subtypes can be homopentamers such as the α7 and α9 or heteropentamers composed of a combination of α (α2–α10) and β (β2–β4) subunits such as the α3β2 and α3β4 subtypes 1,2 . nAChRs are associated with Alzheimer’s, Parkinson’s and schizophre- nia 3,5–7 and the therapeutic potential of nAChRs rests on the ability to develop subtype selective modulators that can defne the relative role of each of the diferent subtypes in normal and disease processes 8 . Many of the available plant and animal toxins have naturally engineered specifcity for the α1β1γδ/ε (muscle), α7 and α3β2 (neuronal) subtypes 9–11 . Tis provided opportunities for several detailed investigations into the ligand recognition and selectivity mechanisms at these subtypes, providing the framework required for the rational development of therapeutics 12–18 . In comparison, such detailed structural and functional characterisations of the α3β4 activity are currently lacking. Primarily due to the small number of α3β4 specifc peptides (Table 1). α3β4 is the predominant nAChR subtype in the autonomic nervous system, contributing to the “reward” sensation associated with nico- tine addiction and drug abuse as well as the development and progression of lung cancer 19–21 . Tus antagonists of the α3β4 nAChR may have anti-addictive and anti-cancer potential. α-Conotoxins are a large family of disulfde rich peptide antagonists of the nAChRs isolated from the venom of marine cone snails 11,22 . Over ffy α-conotoxins have been isolated and characterised to date 11 , including a small number with α3β4 activity (Table 1). To broaden our understanding of nAChR pharmacology, we used α-conotoxin LsIA to identify the minimum structural requirements for α-conotoxin activity at human α3β4 nAChR. Native LsIA is an equipotent antagonist of the human α7 and rat α3β2 but inactive at α3β4 nAChRs 23 despite relatively high sequence identity to α-conotoxins with activity at the α3β4 nAChRs 23–32 (Table 1). Using a co-crystal structure of LsIA and Lymnaea stagnalis acetylcholine binding protein (AChBP) to guide mutational iMB centre for Pain Research, institute for Molecular Bioscience, the Universi ty of Queensland, St. Lucia, Queensland 4072, Australia. Correspondence and requests for materials should be addressed to R.L. (email: r.lewis@ imb.uq.edu.au) Received: 03 October 2016 Accepted: 01 March 2017 Published: 31 March 2017 opeN