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.

Marios Zouridakis,Socrates Tzartos,Athanasios Papakyriakou,Igor E Kasheverov,Victor Tsetlin,Petros Giastas,Igor A Ivanov

doi:10.3389/fphar.2019.00474

Abstract

The α9 subunit of nicotinic acetylcholine receptors (nAChRs) exists mainly in heteropentameric assemblies with α10. Accumulating data indicate the presence of three different binding sites in α9α10 nAChRs: the α9(+)/α9(−), the α9(+)/α10(−), and the α10(+)/α9(−). The major role of the principal (+) side of the extracellular domain (ECD) of α9 subunit in binding of the antagonists methyllylcaconitine and α-bungarotoxin was shown previously by the crystal structures of the monomeric α9-ECD with these molecules. Here we present the 2.26-Å resolution crystal structure of α9-ECD in complex with α-conotoxin (α-Ctx) RgIA, a potential drug for chronic pain, the first structure reported for a complex between an nAChR domain and an α-Ctx. Superposition of this structure with those of other α-Ctxs bound to the homologous pentameric acetylcholine binding proteins revealed significant similarities in the orientation of bound conotoxins, despite the monomeric state of the α9-ECD. In addition, ligand-binding studies calculated a binding affinity of RgIA to the α9-ECD at the low micromolar range. Given the high identity between α9 and α10 ECDs, particularly at their (+) sides, the presented structure was used as template for molecular dynamics simulations of the ECDs of the human α9α10 nAChR in pentameric assemblies. Our results support a favorable binding of RgIA at α9(+)/α9(−) or α10(+)/α9(−) rather than the α9(+)/α10(−) interface, in accordance with previous mutational and functional data.

Highlights

Nicotinic acetylcholine receptors are the prototypic members of the Cys-loop family of pentameric ligand-gated ion channels, including the 5-HT3, GABAA, and glycine receptors (Lester et al, 2004; Sine and Engel, 2006; Albuquerque et al, 2009; Nemecz et al, 2016)
Whereas the structure of the α9-extracellular domain (ECD) in this complex was very similar to the previously determined structure of the apo α9-ECD (Zouridakis et al, 2014), presenting a root mean square deviation (RMSD) value of 0.497 Å for their paired Cα atoms, the crystal structure of RgIA bound to α9-ECD presented an RMSD value for Cα atoms of 1.905 Å compared to its NMR structure in solution (PDB ID: 2JUT) (Ellison et al, 2008)
In the crystal structure of RgIA, an intramolecular salt bridge between Asp5 and Arg7 was observed (Figure 1D), missing from its NMR structure. To investigate whether these observed conformational changes of RgIA upon binding to α9-ECD have been reported in other cases, we sought for examples involving α-Ctxs

Summary

Introduction

Nicotinic acetylcholine receptors are the prototypic members of the Cys-loop family of pentameric ligand-gated ion channels, including the 5-HT3, GABAA, and glycine receptors (Lester et al, 2004; Sine and Engel, 2006; Albuquerque et al, 2009; Nemecz et al, 2016). Due to the high similarity in the orthosteric ligand-binding site of neuronal nAChRs, the development of drugs targeting a distinct nAChR subtype is a very challenging task, requiring detailed structural information. This site consists of loops A, B, and C of the principal (+) side of the ECD of an α subunit and of loops D, E, and F of the complementary (−) side of the ECD of the adjacent α or β subunit (Brejc et al, 2001; Unwin, 2005). Whereas the highly conserved (+) side of the ligand-binding site seems to play an important role in the orientation of the bound ligand (Dellisanti et al, 2007; Zouridakis et al, 2014), it is the less conserved (−) side that determines the selectivity on a specific nAChR subtype (Rucktooa et al, 2009; Bourne et al, 2015; Giastas et al, 2018)

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Discussion

Conclusion