Site-specific post-translational modifications regulate the binding affinity of Conus amadis α-conotoxin Am2005 to...

Post-translational modifications (PTMs) play a pivotal role in diversifying the structure and function of peptide toxins from marine cone snails, which have proven applications in the treatment of neuropathic pain. Analysis of crude venom from Conus amadis using transcriptomic peptide sequence combined with tandem mass spectrometry revealed site-specific PTMs in α-conotoxin Am2005. Six distinct variants were identified, differing in PTMs, such as 4-trans-hydroxylation of proline, γ-carboxylation of glutamic acid, and C-terminal extension by an arginine residue (Am2161). This report aims to elucidate the significance of these site-specific PTMs on 3D conformation and binding affinity of Am2005 toward Aplysia californica acetylcholine-binding protein (Ac-AChBP), a useful model to study nicotinic acetylcholine receptors (nAChRs). Conserved disulfide core of Am2005 with α-conotoxin LvIA, along with the availability of co-crystal structure of α-conotoxin LvIA with Ac-AChBP, prompted the use of computational methods for predicting 3D structure by homology modelling and calculating binding affinity through peptide-protein docking methods. Site-specific PTMs affect the 3D structure by altering local hydrogen-bonding networks, thereby changing the orientation of side chains in 3D space while retaining canonical globular fold with a helical motif. [P2O,P15O,E16γ]Am2161 exhibits a 2-fold higher binding affinity to Ac-AChBP and MD simulations indicates [P2O,P15O,E16γ]Am2161/[P2O,P8O,E16γ]Am2161 have relatively higher hydrogen bonds with Ac-AChBP. Furthermore, peptides with similar binding affinities differed 3-fold in hydrophobicity, as revealed by aggregation score calculations, confirming the role of PTMs in modulating the hydrophobic patch. These studies unravel the significance of site-specific PTMs in fine-tuning the affinity of α-conotoxin Am2005 to its molecular target, which may aid prey capture.