Single disulfide conopeptides Structure and allosteric activity of a single disulfide conopeptide from Conus zonatus at human α3 β4 and α7 nicotinic acetylcholine receptors. Madhan Kumar Mohan 1 , Nikita Abraham 2 , Rajesh R P 3 , Benjamin Franklin Jayaseelan 4 , Lotten Ragnarsson 2 , Richard J. Lewis 2 , Siddhartha P. Sarma 1 From the 1 Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka - 560012, INDIA. 2 Institute for Molecular Bioscience, Queensland Bioscience Precinct (Building 80), The University of Queensland, 306 Carmody Road, St Lucia Qld 4072, Australia. 3 Sathyabama Institute of Science and Technology, Jeppiaar Nagar, Rajiv Gandhi Salai, Chennai - 600119, Tamil Nadu, India. 4 Bombay Natural History Society, Hornbill House, Dr. Salim Ali Chowk, Mumbai 400 001, Maharashtra, India. Running title: Single disulfide conopeptides To whom correspondence should be addressed: Siddhartha P. Sarma, Tel: +91 80 22933454, Fax: +91 80 2360 0535, sidd@iisc.ac.in.; Richard J. Lewis, Tel: +61 7 334 62984, r.lewis@uq.edu.au Keywords: nAChR anatagonists, single-disulfide conopeptides, allosteric modulation, conformational exchange, Proline cis - trans isomerism, NMR, conotoxin, nicotinic acetylcholine receptor, neurotoxin, Czon1107 ABSTRACT Conopeptides are neurotoxic peptides in the venom of marine cone snails and have broad therapeutic potential for managing pain and other conditions. Here, we identified the single-disulfide peptides Czon1107 and Cca1669 from the venoms of Conus zonatus and Conus caracteristicus, respectively. We observed that Czon1107 strongly inhibits the human α3 β4 (IC50 15.7 ± 3.0 μM) and α7 (IC50 77.1 ± 0.05 μM) nicotinic acetylcholine receptor (nAChR) subtypes, but the activity of Cca1669 remains to be identified. Czon1107 acted at a site distinct from the orthosteric receptor site. Solution NMR experiments revealed that Czon1107 exists in equi- librium between conformational states that are the result of a key Ser 4 —Pro 5 cis—trans isomerization. Moreover, we found that the X-Pro amide bonds in the inter-cysteine loop are rigidly constrained to cis conformations. Structure–activity experiments of Czon1107 and its variants at positions P5 and P7 re- vealed that the conformation around the X-Pro bonds (cis—trans) plays an important role in receptor sub- type selectivity. The cis conformation at the Cys 6 — Pro 7 peptide bond was essential for α3 β4 nAChR subtype allosteric selectivity. In summary, we have identified an unique single disulfide conopeptide with a non-competitive, potentially allosteric in- hibitory mechanism at the nAChRs. The small size and rigidity of the Czon1107 peptide could provide a scaffold for rational drug design strategies for al- losteric nAChR modulation. This new paradigm in the “conotoxinomic” structure–function space pro- vides an impetus to screen venom from other Conus species for similar, short bioactive peptides that al- losterically modulate ligand-gated receptor function. Conotoxins and conopeptides are neurotoxic pep- tides found in the venom of predatory marine cone snails. The peptide components in the venom are broadly classified as disulfide-rich or disulfide-poor, wherein the former are referred to as conotoxins (≥ 2 S-S) and the latter as conopeptides (≤ 1 S-S). The current system of classification has placed the conotoxins in 29 gene superfamilies that are repre- sentative of 30 cysteine frameworks(1). In contrast, 1 https://www.jbc.org/cgi/doi/10.1074/jbc.RA119.012098 The latest version is at JBC Papers in Press. Published on March 31, 2020 as Manuscript RA119.012098 by guest on July 24, 2020 http://www.jbc.org/ Downloaded from by guest on July 24, 2020 http://www.jbc.org/ Downloaded from by guest on July 24, 2020 http://www.jbc.org/ Downloaded from