EXPRESSION AND STRUCTURE OF THE ACETYLCHOLINE RECEPTOR

我们的长期目标是阐明潜在的分子机制 利用肌肉的配体-神经递质受体相互作用 以烟碱型乙酰胆碱受体(AChR)为模型。尽管AChR是 近20年前首次提纯和克隆，详细的结构 关于这种蛋白质的信息仍然非常有限。这主要是因为 膜蛋白结晶的内在困难。因此，或Main 目的是表达大量的可溶性N-末端 AChRα和Delta亚基的胞外结构域，以及 确定它们的三维结构。这个域，在Alpha中 亚基，几乎由亚基质量的一半组成，并含有结合 α-银环蛇毒素(α-BuTx)的位置以及所谓的主要 免疫原区(MIR)。这个区域是自身免疫的靶点。 重症肌无力患者的抗体。此外，此域还包含 用于与增量亚单位组装以形成α-的结构信息 能与胆碱能配体结合的Delta杂二聚体，如 乙酰胆碱(ACh)和D-管胡萝卜碱(DTC)。这样做的具体目的是 建议有：1)确定阿尔法上的最小可溶序列 可折叠形成α-但结合位点的亚基和MIR；2)到 定义阿尔法和域亚单位上的最小结构域，可以形成 一种具有高亲和力ACh结合部位的可溶性异二聚体。适当地 折叠的α-N-末端结构域和α-三角洲杂二聚体将是 在酵母中以分泌蛋白的形式表达，纯化至均一；3) 确定α胞外区的3D溶液结构 在自由形式和与α-BuTx的络合物中使用多维核磁共振。 此外，α-三角洲杂二聚体上的结构元素与 ACh和DTC将使用转移的NOE来描绘；4)结晶 α-异二聚体-ACh复合体和MIR-自身免疫抗体复合体 为了用X-射线衍射法研究它们的结构。 这个项目的完成将使我们能够解决长期寻求的问题 AChR的配体结合部位的结构。它还将提供 洞察决定功能的常见结构元素 其他配体门控离子通道，如GABA、甘氨酸和5-羟色胺/3 感受器。因为这些蛋白质在致病机制中起着重要作用。 疼痛、精神疾病和其他常见疾病，如癫痫和 中风，有关其结构的信息对于合理的药物是必不可少的 设计更具选择性的治疗剂。最后，要采用的技术 通过这项工作开发的应该有助于未来对 这些和其他配基门控离子通道。

Our long-term goal is to elucidate the molecular mechanisms underlying ligand-neurotransmitter receptor interactions utilizing the muscle nicotinic acetylcholine receptor (AChR) as a model. Although the AChR was first purified and cloned nearly 20 years ago, detailed structural information of this protein is still very limited. This is mainly due to the inherent difficulty of crystallizing membrane proteins. Hence, or main objective is to express large quantities of soluble, N-terminal extracellular domains of the AChR alpha and delta subunits, and to determine their 3-dimensional structure. This domain, in the alpha subunit, comprises almost half the subunit mass and contains the binding site for alpha-bungarotoxin (alpha-BuTx) as well as the so-called major immunogenic region (MIR). This region is the target of autoimmune antibodies in myasthenia gravis. In addition, this domain contains the structural information for assembly with the delta subunit to form alpha- delta heterodimers which can bind cholinergic ligands, such as acetylcholine (ACh) and d-tubocurarine (dTC). The specific aims of this proposal are: 1) to identify a minimal soluble sequence on the alpha subunit that can fold to form an alpha-BuT binding site and the MIR; 2) to define the minimal domains on the alpha and domain subunits that can form a soluble heterodimer with a high affinity ACh-binding site. Properly folded alpha N-terminal domain and the alphadelta heterodimer will be expressed as secretory proteins in yeast, and purified to homogeneity; 3) to determine the 3D solution structure of the alpha extracellular domain in free form and in complex with alpha-BuTx using multi-dimensional NMR. Also, structural elements on the alphadelta heterodimer interaction with ACh and dTC will be delineated using transferred NOE; 4) to crystalize the alphadelta heterodimer-ACh complex and the MIR-autoimmune antibody complex in order to study their structure using x-ray diffraction methods. Completion of this project will allow us to solve the long-sought structure of the ligand-binding sites of the AChR. It will also provide insight into the common structural elements that determine the function of other ligand-gated ion channels, such as the GABA, glycine and 5-HT/3 receptors. As these proteins play important roles in the pathogenesis of pain, mental illness and other common disorders such as epilepsy and stroke, information on their structure is essential for the rational drug design for more selective therapeutic agents. Finally, techniques to be developed through this work should facilitate future structural studies of these and other ligand-gated ion channels.