Targeting the Homomeric α7 Nicotinic Receptor and Acetylcholine Binding Protein with Lophotoxin by X‐ray Crystallography and Mass Spectrometry

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Lophotoxin is a member of a cyclic diterpenoid family of toxins, devoid of nitrogen, isolated from soft coral Lophogorgia. Previous work suggested irreversible binding of lophotoxin to nicotinic acetylcholine receptors (nAChRs), utilizing electric organs of Torpedo californica; Inhibition is thought to be mediated by an electrophilic addition of the toxin with a hydroxyl of a conserved tyrosine (Tyr 190) of this family of receptors. According to molecular modeling, an alpha‐beta unsaturated carbon in lophotoxin forms a covalent bond with a tyrosine hydroxyl group that will irreversibly inhibit nAChRs. Binding of this unique family of cyclic diterpinoid terpene toxins is associated agonist site of the nAChR, since binding competitively inhibited by reversible agonists, such as epibatidine, or by competitive antagonists, such as MLA. To investigate receptor interactions of lophotoxin with neuronal nicotinic acetylcholine receptors (nAChRs) and related pentameric ligand‐gated ion channels, we employed HEK cells transfected with cDNA’s encoding one of two receptor subtypes: α7‐nAChR and 5HT3AR, along with a fluorescent reporter, and a receptor extracellular domain surrogate, the soluble, acetylcholine binding protein (AChBP). We found that lophotoxin not only inhibited α7‐nAChR function and AChBP ligand binding, as expected, but surprisingly also the function of the serotonin gated ion channel (5HT3A). X‐ray crystallographic studies show the bound lophotoxin to reside in the interfacial site underneath a partially closed C‐loop. Mass spectrometry of the linked peptides show a covalent attachment to peptides bordering the C‐loop bearing a covalent linkage to the tyrosine in the C‐loop. Hence, the binding orientation and atomic residues involved in this marine toxin can be delineated through detailed protein chemistry and crystallographic resolution of the structure. The irreversible binding mode of interaction can serve as a model for potential irreversible inhibitors. Lophotoxin’s irreversible action provides a means for longer term modulation of α7receptor signaling.Support or Funding InformationThe Berkeley Center for Structural Biology is supported in part by the National Institutes of Health, National Institute of General Medical Sciences, and the Howard Hughes Medical Institute. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE‐AC02‐05CH11231. The Pilatus detector was funded under NIH grant S10OD021832. The ALS‐ENABLE beamlines are supported in part by the National Institutes of Health, National Institute of General Medical Sciences, grant P30 GM124169