α1-Fangs: Protein Ligands Selective For The α-Bungarotoxin Site Of The α1-Nicotinic Acetylcholine Receptor
Department
neurobiology
Document Type
Article
Abstract
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 α1 nicotinic subunit (α211). This library yielded 270000 independent potential protein binding ligands. The lead sequence, α1-FANG1, represented 25% of all library sequences, showed the highest-affinity binding, and competed with α-bungarotoxin (α-Btx). To improve this clone, a new library based on α1-FANG1 was subjected to heat, protease, binding, off-rate selective pressures, and point mutations. This resulted in α1-FANG2 and α1-FANG3. These proteins bind α211 with K D values of 3.5 nM and 670 pM, respectively, compete with α-Btx, and show improved subunit specificity. α1-FANG3 is thermostable (T m = 62 °C) with a 6 kcal/mol improvement in folding free energy compared with that of the parent α1-FANG1. α1-FANG3 competes directly with the α-Btx binding site of intact neuromuscular heteropentamers [(α1) 2 β1γδ] 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.
Publication Date
9-21-2018
Publication Title
ACS Chemical Biology
ISSN
15548929
Volume
13
Issue
9
First Page
2568
Last Page
2576
Digital Object Identifier (DOI)
10.1021/acschembio.8b00513
Recommended Citation
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., "α1-Fangs: Protein Ligands Selective For The α-Bungarotoxin Site Of The α1-Nicotinic Acetylcholine Receptor" (2018). Translational Neuroscience. 229.
https://scholar.barrowneuro.org/neurobiology/229