Role of Cys_I–Cys_III Disulfide Bond on the Structure and Activity of α-Conotoxins at Human Neuronal Nicotinic Acetylcholine Receptors

Abstract

α-Conotoxins preferentially antagonize muscle and neuronal nicotinic acetylcholine receptors (nAChRs). Native α-conotoxins have two disulfide links, CI–CIII and CII–CIV, and owing to the inherent properties of disulfide bonds, α-conotoxins have been systematically engineered to improve their chemical and biological properties. In this study, we explored the possibility of simplifying the disulfide framework of α-conotoxins Vc1.1, BuIA, ImI, and AuIB, by introducing [C2H,C8F] modification to the CI–CIII bond. We therefore explored the possibility of using hydrophobic packing of standard amino acid side chains to replace disulfide bonds as an alternative strategy to nonnatural amino acid cross-links. The impact of CI–CIII disulfide bond replacement on the conformation of the α-conotoxins was investigated using molecular dynamics (MD) simulations and nuclear magnetic resonance chemical shift index study. Two-electrode voltage clamp techniques and MD simulations were used to study the impact of disulfide bond deletion on the activities of the peptides at human neuronal nAChRs. All disulfide-deleted variants except ImI[C2H,C8F] had reduced potency for inhibiting nAChRs. Our results suggest that the CI–CIII disulfide bond is important to stabilize the secondary structure of α-conotoxins as well as their interaction with neuronal nAChR targets. Results from this study enrich our understanding of the function of the CI–CIII disulfide bond and are useful in guiding future structural engineering of the α-conotoxins.

Cysteines and Disulfide Bonds as Structure-Forming Units: Insights From Different Domains of Life and the Potential for Characterization by NMR

“…The impact of deletion of disulfide bonds on the activity of α-conotoxins (two disulfide bonds) at human neuronal nicotinic acetylcholine receptors was studied employing NMR, Molecular Dynamics simulations and voltage-clamp techniques (Tabassum et al, 2017). The data supports the notion that the two disulfide bonds have been selectively conserved to create and stabilize a structural scaffold optimized for receptor binding.…”

ConoMode, a database for conopeptide binding modes

“…Currently, a total of 37 modes of binding between conotoxins and nAChRs or ion channels are exhibited on the ConoMode website. Among them, 28 were derived from our previously published studies in recent years ( 33–40 ). These 28 structures were determined using computational modeling strategies, including homology modeling, docking and molecular dynamics simulations and the other 9 were derived from crystal complex structures obtained from the PDB (IDs: 4EZ1, 5CO5, 2C9T, 5XGL, 5JME, 2BR8, 2UZ6, 2BYP, 5T90, 6J8E) ( 10–17 , 23 , 28 ).…”

“…Recently, more than 50 computational models between conotoxins and their targeting ion channels were published, among which more than 20 were published by Yu et al . ( 33–40 ). Most of these models were supported by their ability to explain results from mutagenesis studies.…”

A Novel Natural Influenza A H1N1 Virus Neuraminidase Inhibitory Peptide Derived from Cod Skin Hydrolysates and Its Antiviral Mechanism

“…The initial model was minimized and refined through molecular dynamics (MD) simulations with the Amber 16 package and ff14SB force field [ 39 ]. The system was minimized and equilibrated according the previous method [ 40 ]. The SHAKE algorithm was used for the MD simulations [ 41 ].…”