Structure and allosteric activity of a single-disulfide conopeptide from Conus zonatus at human α3β4 and α7 nicotinic acetylcholine receptors

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 α3β4 (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 equilibrium between conformational states that are the result of a key Ser4–Pro5cis-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 revealed that the conformation around the X-Pro bonds (cis-trans) plays an important role in receptor subtype selectivity. The cis conformation at the Cys6–Pro7 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 Conus species for similar, short bioactive peptides that allosterically modulate ligand-gated receptor function.

Single-Disulfide Conopeptide Czon1107, an Allosteric Antagonist of the Human α3β4 Nicotinic Acetylcholine Receptor

“…In order to identify the residues involved in the interaction between hα3β4 nAChR and conopeptide Czon1107, an alanine (Ala)-scan mutation was carried out on Czon1107. The activity of the synthesized Czon1107 Ala analogues at 10 µM (close to the reported IC 50 of Czon1107 at hα3β4 nAChR [ 28 ]) was determined on ACh-evoked currents at hα3β4 nAChRs ( Figure 3 and Figure S30 ). All peptides inhibited hα3β4-mediated ACh currents in a reversible manner.…”

“…Mutational analysis is widely used to study the relationship between the sequence and structure of peptides [ 28 , 31 ]. In order to identify the residues involved in the interaction between hα3β4 nAChR and conopeptide Czon1107, an alanine (Ala)-scan mutation was carried out on Czon1107.…”

“…In addition to the hα3β4 nAChRs, Czon1107 inhibited nicotine-evoked currents at hα7 nAChRs (IC 50 of 77.2 µM) [ 28 ]. A comparison between hα3β4 and hα7 putatively suggested that Czon1107 residue F2 likely forms a hydrophobic interaction with the hydrophobic P264 residue of hα3β4 nAChR compared to the corresponding hydrophilic and charged D266 residue of hα7 ( Figure S31 ).…”

“…There was a 3 min washout time between perfusions. All peptides were tested at 10 µM, close to the reported IC 50 of wild-type Czon1107 at hα3β4 nAChRs [ 28 ]. The peptide solutions were prepared in ND96 + 0.1% FBS.…”

“…The C. zonatus Czon1107 only has a single disulfide bond and in fluorescence bioassays on heterologous hα3β4 nAChRs expressed in SH-SY5Y cells, Czon1107 non-competitively inhibits nicotine-evoked currents with a half-maximal inhibitory concentration (IC 50 ) of 15.7 µM [ 28 ]. However, the allosteric mechanism of Czon1107 inhibition has not been investigated.…”