α1-FANGs: Protein Ligands Selective for the α-Bungarotoxin Site of the α1-Nicotinic Acetylcholine Receptor.
α1-FANGs: Protein Ligands Selective for the α-Bungarotoxin Site of the α1-Nicotinic Acetylcholine Receptor.
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α1-FANGs：对α1-烟碱乙酰胆碱受体的α-金环蛇毒素位点选择性的蛋白质配体。
DOI：
10.1021/acschembio.8b00513
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发表时间：
2018
影响因子：
4
通讯作者：
Roberts,RichardW
中科院分区：
文献类型：
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作者：
Nichols,AaronL;Noridomi,Kaori;Hughes,ChristopherR;Jalali-Yazdi,Farzad;Eaton,JBrek;Lai,LanHuong;Advani,Gaurav;Lukas,RonaldJ;Lester,HenryA;Chen,Lin;Roberts,RichardW
烟碱型乙酰胆碱受体（nAChRs）是一种五聚体配体门控离子通道，在神经元和神经肌肉的信号转导中发挥重要作用。在这里，我们已经开发了FANG配体，纤连蛋白抗体模拟烟碱乙酰胆碱受体产生的配体，使用mRNA展示。我们构建了一个1万亿成员的主要e10 FnIII文库，以靶向稳定的α1烟碱亚基（α211）。该文库产生了270000个独立的潜在蛋白结合配体。前导序列α1-FANG 1占所有文库序列的25%，显示出最高的亲和力结合，并与α-银环蛇毒素（α-Btx）竞争。为了改进该克隆，对基于α1-FANG 1的新文库进行热、蛋白酶、结合、解离速率选择压力和点突变。这导致α1-FANG 2和α1-FANG 3。这些蛋白结合α211的KD值分别为3.5 nM和670 pM，与α-Btx竞争，并显示出改善的亚基特异性。α1-FANG 3是热稳定的（Tm= 62 °C），与母体α1-FANG 1相比，折叠自由能提高了6 kcal/mol。α1-FANG 3直接与哺乳动物培养细胞膜和表达这些nAChR的非洲爪蟾细胞中完整神经肌肉异五聚体[（α1）2β1γδ]的α-Btx结合位点竞争。这项工作表明，针对五聚体的单体胞外结构域的mRNA展示具有选择在单体和五聚体背景下结合该亚基的配体的能力。总的来说，我们的工作提供了一种途径来创建一个新的稳定，行为良好的蛋白质家族，专门针对这个重要的受体家族。
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 withKDvalues 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 inXenopus laevisoocytes 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.