Rigidity of loop 1 contributes to equipotency of globular and ribbon isomers of \u03b1-conotoxin AusIA

Thao N T Ho,Nikita Abraham,Richard J Lewis

doi:10.1038/s41598-021-01277-4

Abstract

α-Conotoxins are small disulfide-rich peptides targeting nicotinic acetylcholine receptors (nAChRs) characterised by a CICII-Xm-CIII-Xn-CIV framework that invariably adopt the native globular conformations which is typically most potent. α-Conotoxins are divided into several structural subgroups based on the number of residues within the two loops braced by the disulfide bonds (m/n), with the 4/7 and 4/3 subgroups dominating. AusIA is a relatively rare α5/5-conotoxin isolated from the venom of Conus australis. Surprisingly, the ribbon isomer displayed equipotency to the wild-type globular AusIA at human α7-containing nAChR. To understand the molecular basis for equipotency, we determined the co-crystal structures of both isomers at Lymnea stagnalis acetylcholine binding protein. The additional residue in the first loop of AusIA was found to be a critical determinant of equipotency, with 11-fold and 86-fold shifts in potency in favour of globular AusIA over ribbon AusIA observed following deletion of Ala4 or Arg5, respectively. This divergence in the potency between globular AusIA and ribbon AusIA was further enhanced upon truncation of the non-conserved Val at the C-termini. Conversely, equipotency could be replicated in LsIA and TxIA [A10L] following insertion of an Ala in the first loop. These findings provide a new understanding of the role the first loop in ribbon and globular α-conotoxins can play in directing α-conotoxin nAChR pharmacology.

Highlights

NAChRs are prototypical members of the ligand-gated ion channel class of membrane proteins that are found extensively in the central (CNS) and peripheral nervous system (PNS). nicotinic acetylcholine receptors (nAChRs) are interesting therapeutic drug targets associated with the progression of CNS disorders such as epilepsy, Alzheimer’s disease, Parkinson’s disease, and schizophrenia[1,2,3]
Conotoxins are grouped into several “superfamilies” and “families”, which include the enhancers of sodium channels (δ-conotoxin), blockers of sodium, calcium, potassium channels (μ, ω, κA-conotoxin, respectively), as well as competitive and non-competitive antagonists of nAChRs (α- and ψ-conotoxin, respectively)11,12. α-Conotoxins are among the smallest conopeptides (12–20 amino acids) from Conus venoms, with most acting as competitive antagonists of nAChRs
We present the co-crystal structure of gAusIA and rAusIA with Ls-AChBP that allowed us to understand the structural determinants underlying the equipotency at α7 nAChRs of two conformations

Summary

Introduction

NAChRs are prototypical members of the ligand-gated ion channel class of membrane proteins that are found extensively in the central (CNS) and peripheral nervous system (PNS). nAChRs are interesting therapeutic drug targets associated with the progression of CNS disorders such as epilepsy, Alzheimer’s disease, Parkinson’s disease, and schizophrenia[1,2,3]. Α-Conotoxins are among the smallest conopeptides (12–20 amino acids (aa)) from Conus venoms, with most acting as competitive antagonists of nAChRs. Classical α-conotoxins are characterised by a C ICII-Xm-CIII-Xn-CIV framework forming three possible disulfide connectivities: globular (I–III, II–IV), ribbon (I–IV, II–III) and bead (I–II, Scientific Reports | (2021) 11:21928. We present the co-crystal structure of gAusIA and rAusIA with Ls-AChBP that allowed us to understand the structural determinants underlying the equipotency at α7 nAChRs of two conformations. These studies revealed that the additional residue in the first loop of AusIA reduced structural rigidity and was a key contributor to globular and ribbon α-conotoxin equipotency

Methods

Results

Conclusion