α1-FANGs: Protein Ligands Selective for the α-Bungarotoxin Site of the α1-Nicotinic Acetylcholine Receptor

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)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.

Figure 1.

α1-FANG protein sequences and library design. (a) α1-FANG protein sequences. (b) An mRNA display fibronectin selection library contains a promoter sequence (ΔTMV), a constant scaffold with two randomized regions (BC loop and FG loop), and a covalent linkage via puromycin to the nascent fibronectin protein.

Figure 2.

Naïve fibronectin selection against α211. (a) mRNA display selection for the ecto-domain of α211. Binding over background was apparent at pool 3 and reached an appreciable level by pool 6. (b) Radioactive pull down of selected pool 6 fibronectin clones against 100 pmol of α211. We dubbed the clone with the highest level of binding α1-FANG1. (c) Competition of α1-FANG1 with α-Btx. ³⁵S-labeled α1-FANG1 binds beads with immobilized α211; preincubation of α-Btx with α211-immobilized beads causes a decrease in the number of detectable counts of radiolabeled α1-FANG1. (d) α1-FANG1 performance under temperature stress. Pull down of radiolabeled α1-FANG1 against α211 shows α1-FANG1 inactivation at high temperatures.

Figure 3.

Affinity maturation and stabilization of α1-FANG1. (a) Rounds 1–5 of the maturation selection show increased resistance to introduced pressures as the rounds proceed. Rounds 3–5 were exposed to heat in the form of a 65 °C incubation. Rounds 4 and 5 were exposed to Proteinase K (Pro-K). Round 5 included an off-rate competition with α-Btx. (b) Sequence information for clones derived via maturation selection, with α1-FANG2 highlighted. (c) Behavior of individual clones from α1-FANG1-based maturation selection. Individual clones show a range of resistance to the various selective pressures. (d) Comparative pull downs against α211 and the ecto-domain of rat α9 show that α1-FANG2 is highly selective for α211 over α9.

Figure 4.

Melting curves of α1-FANG ligands. (a) Melting curves for α1-FANG1, α1-FANG2, and α1-FANG3 scaffolds at θ₂₂₂. α1-FANG3 shows the most defined transition between two folded states. (b) Folding curves for the clones mentioned above showing the transition from folded to unfolded states.

Figure 5.

Native gel assays show α1-FANG3 binds α211 in solution. (a) Assay of binding of α1-FANG3 and α-Btx to α211. The α211:α-Btx and α211:α1-FANG3 ratios are 1:1 except in lanes 6–9 [α211:α-Btx:α1-FANG3 ratios of 1:1:1 (lane 6), 1:2:1 (lane 7), 1:5:1 (lane 8), and 1:10:1 (lane 9)]. α211 (lane 1), α1-FANG3 (lane 2), and their complex (lane 3) show distinct mobilities under native gel conditions. Heating at 37 °C for 30 min does not affect the α211/α1-FANG3 complex (compare lanes 3 and 4). The α211/α-Btx complex (lane 5) can also be distinguished from the α211/α1-FANG3 complex on the gel. In direct competition for binding to α211, a 1:1 α-Btx:α1-FANG3 ratio gives a gel shift that corresponds to both the α211/α1-FANG3 complex and the α211/α-Btx complex (lane 6). Increasing the α-Btx:α1-FANG ratio upon co-incubation with α211 decreases the intensity of the α211/α1-FANG3 complex band and increases the intensity of the α211/α-Btx complex band (compare lanes 7–9). (b) α1-FANG3 binds α211 specifically with no binding to related family member α9. The α1-FANG3:α9 and α1-FANG3:α211 ratios are 1:1, except in lanes 4 and 7 (2:1). α9 (lane 5) shows no complex with α1-FANG3 (lane 6), while distinct mobilities for α211 (lane 1), α1-FANG3 (lane 2), and their complex (lane 3) as seen in panel a remain.

Figure 6.

α1-FANG3 competes with ¹²⁵I-labeled α-Btx for binding to the intact pentamer in membranes. An α1-FANG3 competition experiment with 10 nM ¹²⁵I-labeled α-Btx is fitted by a K_i of 2.9 nM and maximally inhibits to ~70% of the total signal.

Figure 7.

α1-FANG3 protects against α-Btx inhibition of Ach-induced current in the Xenopus laevis oocyte expression system. (a) Preincubation of oocytes (n = 8) with 200 nM α1-FANG3 for 3 min prior to testing with 10 μM ACh reduces the ACh-induced current by ~5%. Preincubation of the same oocytes with 50 nM α-Btx for 3 min prior to additional testing with 10 μM ACh reduces the ACh-induced current by ~90%. (b) Preincubation of oocytes with 200 nM α1-FANG3 for 5 min and then a mixture of 200 nM α1-FANG3 and 50 nM α-Btx for 3 min shows a protection of ACh-induced current compared to incubations with α-Btx alone. Error bars represent the standard error of the mean (SEM). *** = p value ≤ 0.01