Crystal Structure of the Monomeric Extracellular Domain of α9 Nicotinic Receptor Subunit in Complex With α-Conotoxin RgIA: Molecular Dynamics Insights Into RgIA Binding to α9α10 Nicotinic Receptors.

The alpha 9 subunit of nicotinic acetylcholine receptors (nAChRs) exists mainly in heteropentameric assemblies with alpha 10. Accumulating data indicate the presence of three different binding sites in alpha 9 alpha 10 nAChRs: the alpha 9(C)/alpha 9(-), the alpha 9(C)/alpha 10(-), and the alpha 10(C)/alpha 9(). The major role of the principal (C) side of the extracellular domain (ECD) of alpha 9 subunit in binding of the antagonists methyllylcaconitine and alpha-bungarotoxin was shown previously by the crystal structures of the monomeric alpha 9-ECD with these molecules. Here we present the 2.26-angstrom resolution crystal structure of alpha 9-ECD in complex with alpha-conotoxin (alpha-Ctx) RgIA, a potential drug for chronic pain, the first structure reported for a complex between an nAChR domain and an alpha-Ctx. Superposition of this structure with those of other alpha-Ctxs bound to the homologous pentameric acetylcholine binding proteins revealed significant similarities in the orientation of bound conotoxins, despite the monomeric state of the alpha 9-ECD. In addition, ligand-binding studies calculated a binding affinity of RgIA to the alpha 9-ECD at the low micromolar range. Given the high identity between alpha 9 and alpha 10 ECDs, particularly at their (C) sides, the presented structure was used as template for molecular dynamics simulations of the ECDs of the human alpha 9 alpha 10 nAChR in pentameric assemblies. Our results support a favorable binding of RgIA at alpha 9(C)/alpha 9(-) or alpha 10(C)/alpha 9(-) rather than the alpha 9(C)/alpha 10(-) interface, in accordance with previous mutational and functional data.