Abstract
Nicotinic acetylcholine receptors (nAChR) are therapeutic targets for a range of human diseases. α-Conotoxins are naturally occurring peptide antagonists of nAChRs that have been used as pharmacological probes and investigated as drug leads for nAChR related disorders. However, α-conotoxin interactions have been mostly characterised at the α7 and α3β2 nAChRs, with interactions at other subtypes poorly understood. This study provides novel structural insights into the molecular basis for α-conotoxin activity at α3β4 nAChR, a therapeutic target where subtype specific antagonists have potential to treat nicotine addiction and lung cancer. A co-crystal structure of α-conotoxin LsIA with Lymnaea stagnalis acetylcholine binding protein guided the design and functional characterisations of LsIA analogues that identified the minimum pharmacophore regulating α3β4 antagonism. Interactions of the LsIA R10F with β4 K57 and the conserved –NN– α-conotoxin motif with β4 I77 and I109 conferred α3β4 activity to the otherwise inactive LsIA. Using these structural insights, we designed LsIA analogues with α3β4 activity. This new understanding of the structural basis of protein-protein interactions between α-conotoxins and α3β4 may help rationally guide the development of α3β4 selective antagonists with therapeutic potential.
Highlights
Neuronal nicotinic acetylcholine receptors are ligand gated ion channels involved in the modulation of neurotransmission in the central and peripheral nervous system[1,2,3,4]
LsIA N6 interacts with residues on the plus face of the binding pocket that are highly conserved across the different Nicotinic acetylcholine receptors (nAChR) subtypes
LsIA N12 is reasonably well conserved between LsIA and α-conotoxins active at the α3β4 (Table 1), and is unlikely to be responsible for LsIA inactivity at α3β4 nAChR
Summary
Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand gated ion channels involved in the modulation of neurotransmission in the central and peripheral nervous system[1,2,3,4]. Many of the available plant and animal toxins have naturally engineered specificity for the α1β1γδ/ε(muscle), α7 and α3β2 (neuronal) subtypes[9,10,11] This provided opportunities for several detailed investigations into the ligand recognition and selectivity mechanisms at these subtypes, providing the framework required for the rational development of therapeutics[12,13,14,15,16,17,18]. In comparison, such detailed structural and functional characterisations of the α3β4 activity are currently lacking. Our data reveals a β4 triad comprising K57, I77 and I109 that represents a minimum pharmacophore for α-conotoxins inhibition of α3β4
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