An allosteric binding site of the α7 nicotinic acetylcholine receptor revealed in a humanized acetylcholine-binding protein

Abstract

Nicotinic acetylcholine receptors (nAChRs) belong to the family of pentameric ligand-gated ion channels and mediate fast excitatory transmission in the central and peripheral nervous systems. Among the different existing receptor subtypes, the homomeric α7 nAChR has attracted considerable attention because of its possible implication in several neurological and psychiatric disorders, including cognitive decline associated with Alzheimer's disease or schizophrenia. Allosteric modulators of ligand-gated ion channels are of particular interest as therapeutic agents, as they modulate receptor activity without affecting normal fluctuations of synaptic neurotransmitter release. Here, we used X-ray crystallography and surface plasmon resonance spectroscopy of α7-acetylcholine–binding protein (AChBP), a humanized chimera of a snail AChBP, which has 71% sequence similarity with the extracellular ligand-binding domain of the human α7 nAChR, to investigate the structural determinants of allosteric modulation. We extended previous observations that an allosteric site located in the vestibule of the receptor offers an attractive target for receptor modulation. We introduced seven additional humanizing mutations in the vestibule-located binding site of AChBP to improve its suitability as a model for studying allosteric binding. Using a fragment-based screening approach, we uncovered an allosteric binding site located near the β8–β9 loop, which critically contributes to coupling ligand binding to channel opening in human α7 nAChR. This work expands our understanding of the topology of allosteric binding sites in AChBP and, by extrapolation, in the human α7 nAChR as determined by electrophysiology measurements. Our insights pave the way for drug design strategies targeting nAChRs involved in ion channel–mediated disorders.

Allosteric Agonism of α7 Nicotinic Acetylcholine Receptors: Receptor Modulation Outside the Orthosteric Site

“…The a7 nAChR homology model was constructed without imposing fivefold symmetry, and the MD simulations that were run to refine the homology model enhanced the asymmetry. Considering the asymmetry of the OA sites and the past reports of binding sites in the vestibule of the receptor and its analog a7 AChBP (Spurny et al, 2015;Horenstein et al, 2016;Delbart et al, 2018;Quadri et al, 2019), we docked the ligands into all five interfaces of the receptor (I AB , I BC , I CD , I DE , I EA ) with a grid large enough to cover the vestibular side to ensure that all possible binding sites and multiple subunit configurations were included in our analysis. Docking of 16 diEPP derivatives (Supplemental Fig.…”

Conformational transitions of the serotonin 5-HT3 receptor

“…The protein depicted as ribbon is viewed from inside the vestibule, and the intra-subunit cavity (in the I2 conformation) is represented as a grey surface. Acetate in GLIC, Flurazepam in the bacterial homologue ELIC 69 or a drug fragment in a α 7-AChBP 70 bind to this cavity. A putative rationale for the strong effect of F103 mutants on serotonin EC 50 71 , despite its absence of participation to the binding site, comes from the observation of its concerted motion with that of strands from the neighbouring subunit the motion (around P128 and also around P110, the latter not represented here for clarity).…”

In crystallo-screening for discovery of human norovirus 3C-like protease inhibitors

“…In kinetic assays both J01 and J02 showed inhibitory activity against SV3CP with K i values of 0.37 mM and 0.34 mM, respectively. These values are typical of initial hits in crystallographic fragment-screening studies targetting catalytic- or allosteric-sites of enzymes ( Bauman et al, 2013 , Delbart et al, 2018 , Zhang et al, 2019 ) suggesting that the binding modes we observe in 3CL pro are highly relevant. Since J01 and J02 bind in the active site cleft and maintain the closed conformation of the hairpin, they are good candidates for developing further inhibitors and linking them into a new compound could also improve the bioactivity.…”