Crystal structures of free and antagonist-bound states of human α9 nicotinic receptor extracellular domain

Abstract

We determined the X-ray crystal structures of the extracellular domain (ECD) of the monomeric state of human neuronal α9 nicotinic acetylcholine receptor (nAChR) and of its complexes with the antagonists methyllycaconitine and α-bungarotoxin at resolutions of 1.8 Å, 1.7 Å and 2.7 Å, respectively. The structure of the monomeric α9 ECD superimposed well with the structures of homologous proteins in pentameric assemblies, denoting native folding, despite the absence of a complementary subunit and transmembrane domain. The interaction motifs of both antagonists were similar to those in the complexes with homologous pentameric proteins, thus highlighting the major contribution of the principal side of α9 ECD to their binding. The structures revealed a functionally important β7-β10 strand interaction in α9-containing nAChRs, involving their unique Thr147, a hydration pocket similar to that of mouse α1 ECD and a membrane-facing network coordinated by the invariant Arg210.

Crystal structure of a human neuronal nAChR extracellular domain in pentameric assembly: Ligand-bound α2 homopentamer

“…Interestingly, the above residues at the interface of the ECD and membrane have been shown to play significant role to the signal transduction that leads to the channel gating (10,50,51). However, the extend of the interacting network in the α2-Epi structure is smaller compared with homologous structures (15,21,22), probably signifying the intrinsic flexibility of the membrane-facing loops. …”

“…3C). It is interesting to note here, that the corresponding to α2 Tyr180 residue in the structures of α1 and α9 ECDs, interacts with the equivalent residue to Trp115 of the same subunits (14,15). The advent of the complementary subunit in the α2 ECD structure attracts the side chain of Lys136 toward Tyr180, thus adjusting the relative positions of loops B and E (Fig.…”

“…In this study, we present the crystal structure of the pentameric assembly of the WT α2 nAChR ECD in complex with the agonist epibatidine. To date, only the crystal structures of muscle α1 nAChR ECD in complex with α-bungarotoxin and the neuronal α9 nAChR ECD in its free and antagonist-bound states have been determined (14,15). Both provided invaluable information about the structure of a nAChR ECD, but because of their monomeric state, only the principal face of the ligand binding site was associated with a ligand.…”

“…Recently, we revealed the crystal structures of the ECD of the human α9 nAChR subunit in its free and antagonist-bound states (15), whereas the structure of another ECD nAChR subunit, α1, was also solved earlier by others (14). However, crystal structures of pentameric assemblies of nAChR (ECD or intact) have not been determined so far, since both α1 and α9 ECD structures, although in high resolution, depicted the nAChR ECDs in monomeric forms.…”

“…Because of their considerable homology, especially in their binding sites, designing a novel drug specific for one type of nAChR can be a challenging procedure and requires the addition of more structural information. Following this need, structural studies of the nAChR have been the focus of numerous laboratories, leading to the achievement of many breakthroughs, such as the cryo-electron microscopy structure of the Torpedo nAChR (10) and the X-ray crystal structures of acetylcholine binding proteins (AChBPs; homologs of the ECD of nAChR) (11)(12)(13), mouse muscle-type α1 and human neuronal α9 nAChR ECDs (14,15), GLIC and ELIC (two prokaryotic homologs of pLGICs) (16,17), and two α7 nAChR ECD-AChBP chimeras (18,19). In addition, the structures of other members of the superfamily have recently become available, including that of an invertebrate anionic glutamate receptor (20), the human GABA A β3 (21), the mouse 5-HT 3 receptor (22), the human α3 glycine receptor (23), and the zebrafish α1 glycine receptor (24).…”