AChBP-targeted α-conotoxin correlates distinct binding orientations with nAChR subtype selectivity

Abstract

Neuronal nAChRs are a diverse family of pentameric ion channels with wide distribution throughout cells of the nervous and immune systems. However, the role of specific subtypes in normal and pathological states remains poorly understood due to the lack of selective probes. Here, we used a binding assay based on acetylcholine-binding protein (AChBP), a homolog of the nicotinic acetylcholine ligand-binding domain, to discover a novel α-conotoxin (α-TxIA) in the venom of Conus textile. α-TxIA bound with high affinity to AChBPs from different species and selectively targeted the α3β2 nAChR subtype. A co-crystal structure of Ac-AChBP with the enhanced potency analog TxIA(A10L), revealed a 20° backbone tilt compared to other AChBP–conotoxin complexes. This reorientation was coordinated by a key salt bridge formed between Arg5 (TxIA) and Asp195 (Ac-AChBP). Mutagenesis studies, biochemical assays and electrophysiological recordings directly correlated the interactions observed in the co-crystal structure to binding affinity at AChBP and different nAChR subtypes. Together, these results establish a new pharmacophore for the design of novel subtype-selective ligands with therapeutic potential in nAChR-related diseases.

Insight in nAChR subtype selectivity from AChBP crystal structures

“…The co-crystal structure of the AcAChBP-TxIA(A10L) variant [21] shows that the toxin is bound in a different orientation in the binding site as compared to ImI [22,38] or PnIA(A10L D14K) [20]. The 20° downward tilt of the toxin in the binding site is correlated with a projection of the TxIA(A10L) R5 residue deep into the binding site principal face.…”

“…AChBPs from Lymnaea stagnalis [17], Bulinus truncatus [18] and Aplysia californica [19] share only a 20-24% sequence identity with nAChRs ( Figure 1a) but display a striking structural resemblance with the Torpedo nAChR or mouse α1 nAChR protomer (Figure 1b). Nicotinic ligand binding to these AChBPs was assayed through different methods such as radioligand ( 125 I-αBungarotoxin) displacement, surface plasmon resonance [20][21][22], isothermal titration calorimetry [18] or intrinsic fluorescence quenching [19], and all the AChBPs were found to display a pharmacological profile close to that of the homopentameric α 7 nAChR [18,23].…”

“…It was discovered when using AChBP as bait to fish out novel nAChR subtype selective conotoxins [21]. TxIA binds with high affinity to LsAChBP, followed by the α 3 β 2 and α 7 subtype receptors.…”

Hydrophobic residues at position 10 of α-conotoxin PnIA influence subtype selectivity between α7 and α3β2 neuronal nicotinic acetylcholine receptors

“…Computational docking studies [24] and crystal structures of conotoxin analogues bound to the analogous acetylcholine binding protein (AChBP) have revealed how the toxins and receptors interact, including how the residue at position 10 interacts with the (+)-face of the subunit [25][26][27][28][29][30].…”

An efficient transcriptome analysis pipeline to accelerate venom peptide discovery and characterisation

“…The co-crystallisation of ASIC1a with MitTx, a pain causing Texas coral snake toxin revealed the open state conformation of the channel (Baconguis et al, 2014). Similarly, a number of α-conotoxins including TxIA (Dutertre et al, 2007) and PnIA (Celie et al, 2005), as well as snake toxins such as α-cobratoxin (Bourne et al, 2005), have been used to study binding interactions of nAChRs via its molluscan glial surrogate protein, AChBP (van Dijk et al, 2001). …”