Structural Elucidation of Ivermectin Binding to α7nAChR and the Induced Channel Desensitization

Abstract

The α7 nicotinic acetylcholine receptor (α7nAChR) mediates signaling in the central nervous system and cholinergic anti-inflammatory pathways. Ivermectin is a positive allosteric modulator of a full-length α7nAChR and an agonist of the α7nAChR construct containing transmembrane (TMD) and intracellular (ICD) domains, but structural insights of the binding have not previously been determined. Here, combining nuclear magnetic resonance as a primary experimental tool with Rosetta comparative modeling and molecular dynamics simulations, we have revealed details of ivermectin binding to the α7nAChR TMD + ICD and corresponding structural changes in an ivermectin-induced desensitized state. Ivermectin binding was stabilized predominantly by hydrophobic interactions from interfacial residues between adjacent subunits near the extracellular end of the TMD, where the inter-subunit gap was substantially expanded in comparison to the apo structure. The ion-permeation pathway showed a profile distinctly different from the resting-state profile but similar to profiles of desensitized α7nAChR. The ICD also exhibited structural changes, including reorientation of the MX and h3 helices relative to the channel axis. The resulting structures of the α7nAChR TMD + ICD in complex with ivermectin provide opportunities for discovering new modulators of therapeutic potential and exploring the structural basis of cytoplasmic signaling under different α7nAChR functional states.

Side Groups Convert the α7 Nicotinic Receptor Agonist Ether Quinuclidine into a Type I Positive Allosteric Modulator

“…An initial blind docking of ligand 6 revealed a preference of the docking poses for the interface between the transmembrane domains of each chain (Supplementary Figure S21). This is consistent with the site reported for PNU-120596 in α7 nAChR, and the binding site of ivermectin in both the C. elegans glutamate-gated chloride channel GluClα and α7 nAChR. − This site was then selected for targeted docking, and the centroid of the best five clusters was used as a starting point for molecular dynamics (3 × 500 ns) simulations. After the simulations, the best conformation was selected by root mean square deviation (RMSD) based clustering of the ligand heavy atoms (Figure ).…”

“…On the other hand, the side group of the ligand interacts with Pro240, Leu243, and Ile244 of the (−) M1, with Thr273 and Met276 of the (+) M2, and Ala298, Met301, and Ile302 (Supplementary Figure S22B). Of these interactions, some can be highlighted for their reported importance: Met276 being an amino acid previously reported as critical for the function of other allosteric modulators such as ivermectin and PNU-120596, ,− Leu277 being one of the hydrophobic constriction sites in the receptor pore lining, and Ala280 being one of the amino acid that interacts with the β1−β2 loop after it pivots during activation . The importance of the interactions between the protein and the ligand side group is highlighted by the functional results which showed that smaller substituents, like the bromide in the ligand 5 , fail to potentiate the response, and more polar substituents such as the alcohol group in ligands 8 and 9 have much weaker allosteric effect.…”

“…Met276, a residue in position 15′ of the transmembrane 2 domain, is predicted to interact with the aromatic ring of ligand 6 . This residue is unique to the α7 nAChR sequence and has been shown to be essential for α7-selective PAM I, PAM II, ,, and ivermectin . Furthermore, recent structural insights have shown Met276 as a pivotal contributor to the binding site of PAMs PNU120596 and ivermectin on the transmembrane domain of α7 nAChR.…”

“…This residue is unique to the α7 nAChR sequence and has been shown to be essential for α7-selective PAM I, PAM II, ,, and ivermectin . Furthermore, recent structural insights have shown Met276 as a pivotal contributor to the binding site of PAMs PNU120596 and ivermectin on the transmembrane domain of α7 nAChR. We determined the role of Met276 on ligand 6 potentiating effects by mutating Met276 to leucine, a residue that abolishes α7 PAM activity. − As shown in Figure E, Met276Leu dramatically reduced the ability of ligand 6 to enhance the EC 20 ACh responses of α7 nAChRs.…”

Unconventional PDZ Recognition Revealed in α7 nAChR-PICK1 Complexes

“…This is due to the large radius of gyration (∼60 Å) of a PICK1 dimer, 57 which significantly exceeds the gap between two adjacent subunits of the ICD. 16,40 A future structural study involving α7 nAChR with full-length PICK1, encompassing both the PDZ and BAR domains, may provide additional insights into the interplay between these two proteins.…”