Alanine-Scanning Mutagenesis of α-Conotoxin GI Reveals the Residues Crucial for Activity at the Muscle Acetylcholine Receptor

Abstract

Recently, the muscle-type nicotinic acetylcholine receptors (nAChRs) have been pursued as a potential target of several diseases, including myogenic disorders, muscle dystrophies and myasthenia gravis, etc. α-conotoxin GI isolated from Conus geographus selectively and potently inhibited the muscle-type nAChRs which can be developed as a tool to study them. Herein, alanine scanning mutagenesis was used to reveal the structure–activity relationship (SAR) between GI and mouse α1β1δε nAChRs. The Pro5, Gly8, Arg9, and Tyr11 were proved to be the critical residues for receptor inhibiting as the alanine (Ala) replacement led to a significant potency loss on mouse α1β1δε nAChR. On the contrary, substituting Asn4, His10 and Ser12 with Ala respectively did not affect its activity. Interestingly, the [E1A] GI analogue exhibited a three-fold potency for mouse α1β1δε nAChR, whereas it obviously decreased potency at rat α9α10 nAChR compared to wildtype GI. Molecular dynamic simulations also suggest that loop2 of GI significantly affects the interaction with α1β1δε nAChR, and Tyr11 of GI is a critical residue binding with three hydrophobic amino acids of the δ subunit, including Leu93, Tyr95 and Leu103. Our research elucidates the interaction of GI and mouse α1β1δε nAChR in detail that will help to develop the novel analogues of GI.

Backbone Cyclization Turns a Venom Peptide into a Stable and Equipotent Ligand at Both Muscle and Neuronal Nicotinic Receptors

“…However, the significant decrease in Koff values suggests stronger interaction of the cyclic analogues within one, or both, of the two orthosteric muscle nAChR binding sites. Considering the high sequence homology of α-CIA with α-MI and α-GI, α-CIA is most likely binding at the α-δ interface 35,36 . It has been demonstrated that the ACh binding pocket is mostly composed of hydrophobic residues that interact with residues of the two conotoxin loops formed by the disulfide bridges.…”

Structure-Function Elucidation of a New α-Conotoxin, MilIA, from Conus milneedwardsi

“…It has been shown that the small size and angle of Gly at position 9 is determining for the high affinity [19,23]. More recently, it was shown for toxin GI that the substitution of a Gly for an Ala reduced the affinity for the α/δ binding site by approximately 30-fold [24]. It has been suggested that the Gly has a significant impact on the right-handed helical turn from the α-helix.…”

Advances in Molecular Dynamics Simulations and Enhanced Sampling Methods for the Study of Protein Systems

“…Alanine scanning mutagenesis is another experimental technique that has been computerized to accelerate ‘hotspot’ prediction, mostly at protein-protein interface. This method involved the site-directed mutagenesis of specific residues to alanine to assess the contribution of the amino acid towards protein stability and function [ 103 , 104 , 105 ]. Computational alanine scanning (CAS), a robust alternative to its experimental counterpart, permits the rapid scanning of protein surface for amino acid clusters vital for protein-protein association [ 74 ].…”