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

Abstract

Abstractα-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.

Unravelling the allosteric binding mode of αD-VxXXB at nicotinic acetylcholine receptors

“…Based on previous experimental determinations ( Abraham et al, 2017 ; Ho et al, 2021a ; Ho et al, 2021b ), purified de-tagged Ls -AChBP and synthesized VxXXB-C(21–50) were mixed at a molar ratio of 1:2 at 4°C for 1 h before setting up crystallization trials. Crystals were successfully grown at room temperature using the hanging drop method by mixing protein and reservoir solution composed of 0.91 M lithium chloride, 16% PEG6000 and 0.1 M MES monohydrate pH 6.4 at a ratio 1:1 v/v.…”

Marine Origin Ligands of Nicotinic Receptors: Low Molecular Compounds, Peptides and Proteins for Fundamental Research and Practical Applications

“…In particular, the structure of the complex of this protein with α-conotoxin LsIA resulted in identification of the minimum pharmacophore regulating the α3β4 antagonism [ 131 ]. Crystallization with L. stagnalis AChBP of the two isoforms (globular and ribbon) of the unusual α5/5-conotoxin AusIA, equipotential to the α7 nAChR in combination with the substitutions in the peptide, made it possible to explain this pharmacological feature due to the presence of the fifth amino acid residue in the first loop of this conotoxin, and also to determine the key residues responsible for the α7 selectivity ( Table 2 ) [ 136 ].…”