Structure and Allosteric Activity of a Single-disulfide Conopeptide from at Human α3β4 and α7 Nicotinic Acetylcholine Receptors

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 and , respectively. We observed that Czon1107 strongly inhibits the human α3β4 (IC 15.7 ± 3.0 μm) and α7 (IC 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 equilibrium between conformational states that are the result of a key Ser-Pro isomerization. Moreover, we found that the X-Pro amide bonds in the inter-cysteine loop are rigidly constrained to conformations. Structure-activity experiments of Czon1107 and its variants at positions P5 and P7 revealed that the conformation around the X-Pro bonds () plays an important role in receptor subtype selectivity. The conformation at the Cys-Pro peptide bond was essential for α3β4 nAChR subtype allosteric selectivity. In summary, we have identified a unique single-disulfide conopeptide with a noncompetitive, potentially allosteric inhibitory mechanism at the nAChRs. The small size and rigidity of the Czon1107 peptide could provide a scaffold for rational drug design strategies for allosteric nAChR modulation. This new paradigm in the "conotoxinomic" structure-function space provides an impetus to screen venom from other species for similar, short bioactive peptides that allosterically modulate ligand-gated receptor function.