Beactica AB, Box 567, SE-751 22 Uppsala, Sweden.
Biochemistry. 2010 Sep 21;49(37):8143-54. doi: 10.1021/bi1006354.
The mechanism of agonist interactions with Cys-loop ligand-gated ion channels has been studied using the acetylcholine-binding protein (AChBP) from Lymnaea stagnalis as a model protein and acetylcholine, nicotine, epibatidine, and a series of substituted quinuclidines as ligands. A biosensor-based assay for direct interaction studies of immobilized AChBP and small molecule ligands was developed. It allowed the characterization of the interaction kinetics of the ligands and the structural dynamics of the protein. The interactions with AChBP were very sensitive to variations in the experimental conditions and showed several types of complexities. These could be resolved into two types of ligand-induced secondary effects with different kinetics, representing fast and slow conformational changes. The data could be rationalized in a mechanistic model, and a structural interpretation of the interaction was obtained by molecular modeling involving induced fit and loop flexibility simulations. The data suggest that AChBP exhibits ligand-induced structural dynamics, as expected for the ligand gating mechanism of Cys-loop receptors. It shows that the formation of the initial encounter complex between AChBP and ligands is very rapid, in accordance with the functional characteristics required of neurotransmission. These developed procedures will enable further exploration of the mechanism of Cys-loop receptor function and the identification of specific ligands suitable for pharmacological use.
已使用来自田螺的乙酰胆碱结合蛋白 (AChBP) 作为模型蛋白，并以乙酰胆碱、尼古丁、epibatidine 和一系列取代的奎宁环作为配体，研究了激动剂与 Cys 环配体门控离子通道的相互作用机制。开发了一种基于生物传感器的测定法，用于研究固定化 AChBP 和小分子配体的直接相互作用。它允许对配体的相互作用动力学和蛋白质的结构动力学进行表征。与 AChBP 的相互作用对实验条件的变化非常敏感，并显示出几种类型的复杂性。这些可以分解为两种具有不同动力学的配体诱导的二级效应，代表快速和缓慢的构象变化。可以通过涉及诱导契合和环灵活性模拟的分子建模，将数据合理化到机械模型中，并获得相互作用的结构解释。数据表明，AChBP 表现出配体诱导的结构动力学，这是 Cys 环受体配体门控机制所预期的。它表明 AChBP 与配体之间初始遭遇复合物的形成非常迅速，这与神经传递所需的功能特征一致。这些开发的程序将能够进一步探索 Cys 环受体功能的机制，并确定适合药理学用途的特定配体。
