A Novel Lid-Covering Peptide Inhibitor of Nicotinic Acetylcholine Receptors Derived from αD-Conotoxin GeXXA

Abstract

Nicotinic acetylcholine receptors (nAChRs) play a fundamental role in nervous signal transmission, therefore various antagonists and agonists are highly desired to explore the structure and function of nAChRs. Recently, a novel dimeric αD-conotoxin GeXXA was identified to inhibit nAChRs by binding at the top surface of the receptors, and the monomeric C-terminal domain (CTD) of αD-GeXXA retains some inhibitory activity. In this study, the internal dimeric N-terminal domain (NTD) of this conopeptide was further investigated. We first developed a regio-selective protection strategy to chemically prepare the anti-parallel dimeric NTD, and found that the isolated NTD part of GeXXA possesses the nAChR-inhibitory activity, the subtype-dependence of which implies a preferred binding of NTD to the β subunits of nAChR. Deletion of the NTD N-terminal residues did not affect the activity of NTD, indicating that the N-terminus is not involved in the interaction with nAChRs. By optimizing the sequence of NTD, we obtained a fully active single-chain cyclic NTD, based on which 4 Arg residues were found to interact with nAChRs. These results demonstrate that the NTD part of αD-GeXXA is a "lid-covering" nAChR inhibitor, displaying a novel inhibitory mechanism distinct from other allosteric ligands of nAChRs.

Marine natural products

“…650 Anti-parallel dimeric N-terminal domain peptides related to aD-conotoxin GeXXA (C. generalis) are antagonists of nAChR, acting by a 'lid-covering' mechanism, which is distinct from other neurotoxins that bind to the interface between extracellular domains or are pore blockers. 651 Venom of C. generalis was also the source of O-conotoxin GeXXVIIA which in its native form is a disulde-linked homodimer. 652 The linear peptide of the toxin exhibited potent inhibition of human nAChR.…”

Neuronal Nicotinic Acetylcholine Receptor Modulators from Cone Snails

“…MrIC: MrIC was identified from C. marmoreus using transcriptomic approaches, and has the typical α-conotoxin cysteine framework and three-dimensional structure [ 77 ]. Interestingly, MrIC was shown to be an agonist at the α7 nAChR in the presence of type II nAChR positive allosteric modulator, suggesting that the MrIC had unique receptor state dependence [ 77 , 106 ]. Given that the cysteine framework, disulfide pattern, three-dimensional structure, and potentially the binding mode conform to that of typical α-conotoxins, this unusual behavior is likely to be driven by the primary sequence differences.…”

“…Interestingly, when synthesized as a monomers, the N- and C- termini retained inhibitory activity independent of each other, albeit with reduced potency when compared to the native dimeric form. Based on receptor mutagenesis and systematically truncated analogues, the αD-GeXXA binding mechanism is proposed to involve the two C-termini of the dimer spanning across the top of the extracellular domain of the receptor to interact with residues on the α10 subunits, which results in the N-terminii ‘covering’ the pore of the nAChR [ 106 ]. This is proposed to stabilize the inactive/resting state of the receptor, preventing the global conformational changes that are required for channel activation [ 106 ], thereby explaining its allosteric pharmacology.…”

“…Based on receptor mutagenesis and systematically truncated analogues, the αD-GeXXA binding mechanism is proposed to involve the two C-termini of the dimer spanning across the top of the extracellular domain of the receptor to interact with residues on the α10 subunits, which results in the N-terminii ‘covering’ the pore of the nAChR [ 106 ]. This is proposed to stabilize the inactive/resting state of the receptor, preventing the global conformational changes that are required for channel activation [ 106 ], thereby explaining its allosteric pharmacology. Given the αDs from different species have different nAChR subtype selectivity, identifying the pharmacophores for other αDs could provide novel, allosteric strategies of subtype selective nAChR inhibition.…”

“…However, the size, complexity, and unknown disulfide connectivity of most non-classical α-conotoxins makes them challenging for chemical synthesis. Nevertheless, breakthroughs are being made in this aspect, as evidenced by the work of Yang et al (2017), increasing the potential of these α-conotoxins to contribute to the discovery of new ligand recognitions mechanisms at the nAChRs [ 106 ].…”