3 years ago

[ASAP] a1-FANGs: Protein Ligands Selective for the a-Bungarotoxin Site of the a1-Nicotinic Acetylcholine Receptor

[ASAP] a1-FANGs: Protein Ligands Selective for the a-Bungarotoxin Site of the a1-Nicotinic Acetylcholine Receptor
Richard W. Roberts, Aaron L. Nichols, Ronald J. Lukas, Kaori Noridomi, Lan Huong Lai, J. Brek Eaton, Henry A. Lester, Farzad Jalali-Yazdi, Lin Chen, Christopher R. Hughes, Gaurav Advani
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 KD values of 3.5 nM and 670 pM, respectively, compete with α-Btx, and show improved subunit specificity. α1-FANG3 is thermostable (Tm = 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)2β1γδ] 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.

Publisher URL: http://dx.doi.org/10.1021/acschembio.8b00513

DOI: 10.1021/acschembio.8b00513

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