Soluble, oligomeric, and ligand-binding extracellular domain of the human α7 acetylcholine receptor expressed in yeast -: Replacement of the hydrophobic cysteine loop by the hydrophilic loop of the ACh-binding protein enhances protein solubility
Soluble, oligomeric, and ligand-binding extracellular domain of the human α7 acetylcholine receptor expressed in yeast -: Replacement of the hydrophobic cysteine loop by the hydrophilic loop of the ACh-binding protein enhances protein solubility
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DOI：
10.1074/jbc.m402533200
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发表时间：
2004-09-10
影响因子：
4.8
通讯作者：
Tzartos, SJ
中科院分区：
文献类型：
--
作者：
Avramopoulou, V;Mamalaki, A;Tzartos, SJ
将神经元烟碱乙酰胆碱受体（AChR）α 7亚基的N-末端胞外结构域（ECD;氨基酸1-208）（已知唯一组装为同五聚体的人AChR亚基）在酵母毕赤酵母中表达为糖基化形式，以获得完整五聚体烟碱AChR的胞外部分的天然样模型。这种分子，α 7-ECD，虽然能够结合特异性配体α-银环蛇毒素，主要以微聚集体的形式存在。用丝氨酸取代α 7-ECD中的Cys-116导致微聚集体尺寸减小。产生了第二种突变形式，α 7-ECD（C116 S，Cys-环），其中除了C116 S突变之外，疏水性Cys-环（Cys（128）-Cys（142））被来自蜗牛神经胶质细胞乙酰胆碱结合蛋白的相应亲水性Cys-环取代。该第二种突变蛋白是水溶性的，以中等水平（0.5+/-0.1mg/升）表达，并且具有通过凝胶过滤和电子显微镜研究判断的大约对应于五聚体的大小。它还以相对较高的亲和力（Kd =57 nM）结合I-125-α-银环蛇毒素，该结合被未标记的α-银环蛇毒素、d-筒箭毒碱或尼古丁抑制（分别为Ki =0.8x10（-7）M、Ki = 1x 10（-5）M和Ki =0.9x10（-2）M）。所有三种构建体均以糖基化形式表达，但体外去糖基化降低了异质性，而不影响其配体结合特性。这些结果表明，α 7-ECD（C116 S，Cys环）在巴斯德毕赤酵母中表达为具有接近天然构象的寡聚体（可能是五聚体），并且其去糖基化形式似乎是用于神经递质受体配体结合结构域结构研究的合适起始材料。
The N-terminal extracellular domain (ECD; amino acids 1-208) of the neuronal nicotinic acetylcholine receptor (AChR) alpha7 subunit, the only human AChR subunit known to assemble as a homopentamer, was expressed as a glycosylated form in the yeast Pichia pastoris in order to obtain a native-like model of the extracellular part of an intact pentameric nicotinic AChR. This molecule, alpha7-ECD, although able to bind the specific ligand alpha-bungarotoxin, existed mainly in the form of microaggregates. Substitution of Cys-116 in the alpha7-ECD with serine led to a decrease in microaggregate size. A second mutant form, alpha7-ECD(C116S, Cys-loop), was generated in which, in addition to the C116S mutation, the hydrophobic Cys-loop (Cys(128)-Cys(142)) was replaced by the corresponding hydrophilic Cys-loop from the snail glial cell acetylcholine-binding protein. This second mutant protein was water-soluble, expressed at a moderate level (0.5+/-0.1 mg/liter), and had a size corresponding approximately to a pentamer as judged by gel filtration and electron microscopy studies. It also bound I-125-alpha-bungarotoxin with relatively high affinity (K-d=57 nM), the binding being inhibited by unlabeled alpha-bungarotoxin, d-tubocurarine, or nicotine (K-i=0.8x10(-7) M, K-i=1x10(-5) M, and K-i=0.9x10(-2) M, respectively). All three constructs were expressed as glycosylated forms, but in vitro deglycosylation reduced the heterogeneity without affecting their ligand binding properties. These results show that alpha7-ECD(C116S, Cys-loop) was expressed in P. pastoris as an oligomer (probably a pentamer) with a near native conformation and that its deglycosylated form seems to be suitable starting material for structural studies on the ligand-binding domain of a neurotransmitter receptor.