Î±1-Fangs: Protein Ligands Selective For The Î±-Bungarotoxin Site Of The Î±1-Nicotinic Acetylcholine Receptor
Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that play a central role in neuronal and neuromuscular signal transduction. Here, we have developed FANG ligands, fibronectin antibody-mimetic nicotinic acetylcholine receptor-generated ligands, using mRNA display. We generated a 1 trillion-member primary e10FnIII library to target a stabilized Î±1 nicotinic subunit (Î±211). This library yielded 270000 independent potential protein binding ligands. The lead sequence, Î±1-FANG1, represented 25% of all library sequences, showed the highest-affinity binding, and competed with Î±-bungarotoxin (Î±-Btx). To improve this clone, a new library based on Î±1-FANG1 was subjected to heat, protease, binding, off-rate selective pressures, and point mutations. This resulted in Î±1-FANG2 and Î±1-FANG3. These proteins bind Î±211 with K D values of 3.5 nM and 670 pM, respectively, compete with Î±-Btx, and show improved subunit specificity. Î±1-FANG3 is thermostable (T m = 62 Â°C) with a 6 kcal/mol improvement in folding free energy compared with that of the parent Î±1-FANG1. Î±1-FANG3 competes directly with the Î±-Btx binding site of intact neuromuscular heteropentamers [(Î±1) 2 Î²1Î³Î´] in mammalian culture-derived cellular membranes and in Xenopus laevis oocytes expressing these nAChRs. This work demonstrates that mRNA display against a monomeric ecto-domain of a pentamer has the capability to select ligands that bind that subunit in both a monomeric and a pentameric context. Overall, our work provides a route to creating a new family of stable, well-behaved proteins that specifically target this important receptor family.
ACS Chemical Biology
Digital Object Identifier (DOI)
Nichols, Aaron L.; Noridomi, Kaori; Hughes, Christopher R.; Jalali-Yazdi, Farzad; Eaton, J. Brek; Lai, Lan Huong; Advani, Gaurav; Lukas, Ronald J.; Lester, Henry A.; Chen, Lin; and Roberts, Richard W., "Î±1-Fangs: Protein Ligands Selective For The Î±-Bungarotoxin Site Of The Î±1-Nicotinic Acetylcholine Receptor" (2018). Neurobiology. 229.