Molecular blueprint of allosteric binding sites in a homologue of the agonist-binding domain of the α7 nicotinic acetylcholine receptor

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 112; no. 19; pp. E2543 - E2552
• Main Authors: Spurny, Radovan, Debaveye, Sarah, Farinha, Ana, Veys, Ken, Vos, Ann M., Gossas, Thomas, Atack, John, Bertrand, Sonia, Bertrand, Daniel, Danielson, U. Helena, Tresadern, Gary, Ulens, Chris
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 12.05.2015; National Acad Sciences
• Series: PNAS Plus
• Subjects: Allosteric Regulation; Allosteric Site; alpha7 Nicotinic Acetylcholine Receptor - metabolism; Animals; Biological Sciences; Carbon - chemistry; Crystallography, X-Ray; Humans; Ligand-Gated Ion Channels - metabolism; Ligands; Models, Molecular; Mutagenesis; Oocytes - metabolism; PNAS Plus; Protein Binding; Protein Structure, Tertiary; Receptors, Nicotinic - metabolism; Surface Plasmon Resonance; Torpedo; X-Ray Diffraction; Xenopus; X-ray crystallography; Cys-loop receptor; fragment screen; allosteric modulator; ligand-gated ion channel
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1418289112

The α7 nicotinic acetylcholine receptor (nAChR) belongs to the family of pentameric ligand-gated ion channels and is involved in fast synaptic signaling. In this study, we take advantage of a recently identified chimera of the extracellular domain of the native α7 nicotinic acetylcholine receptor and acetylcholine binding protein, termed α7-AChBP. This chimeric receptor was used to conduct an innovative fragment-library screening in combination with X-ray crystallography to identify allosteric binding sites. One allosteric site is surface-exposed and is located near the N-terminal α-helix of the extracellular domain. Ligand binding at this site causes a conformational change of the α-helix as the fragment wedges between the α-helix and a loop homologous to the main immunogenic region of the muscle α1 subunit. A second site is located in the vestibule of the receptor, in a preexisting intrasubunit pocket opposite the agonist binding site and corresponds to a previously identified site involved in positive allosteric modulation of the bacterial homolog ELIC. A third site is located at a pocket right below the agonist binding site. Using electrophysiological recordings on the human α7 nAChR we demonstrate that the identified fragments, which bind at these sites, can modulate receptor activation. This work presents a structural framework for different allosteric binding sites in the α7 nAChR and paves the way for future development of novel allosteric modulators with therapeutic potential. Significance In this study we take advantage of a recently described chimera of the α7 nicotinic acetylcholine receptor (nAChR) and acetylcholine binding protein (AChBP), termed α7-AChBP. To date, more than 70 crystal structures have been determined for AChBP in complex with ligands that occupy the orthosteric binding site. Here, we use an innovative screening strategy to discover molecular fragments that occupy allosteric binding sites. In combination with X-ray crystallography we determine a molecular blueprint of three different allosteric sites in α7-AChBP. Using electrophysiological recordings on the human α7 nAChR we demonstrate that each of the three sites is involved in allosteric modulation of the receptor. Our study contributes to understanding the sites of allosteric binding in ion channels.