Properties of Specific Alcohol Binding Sites

特定醇结合位点的特性

• 批准号: 6603848
• 负责人: JAMES Robert TRUDELL
• 金额: $ 23.55万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6603848
• 关键词: GABA receptor; acetylcholine; alcohols; binding sites; chemical models; cysteine; disulfide bond; glycine receptors; membrane channels; model design /development; molecular biology information system; molecular dynamics; nicotinic receptors; point mutation; receptor binding; serotonin receptor

DESCRIPTION (provided by applicant): Alcohol abuse and alcoholism are major health problems. It is likely that a solution to these problems will require an understanding of the effects of alcohol on specific ion channels and the kinases that modulate them. Specific binding sites for alcohols have recently been described in the transmembrane domain of the superfamily of glycine, GABA, nicotinic acetylcholine, and 5-HT3 receptors. Our hypothesis is that alcohols bind within cavities that are bounded by transmembrane segments of these receptors. Our goal is to define the properties of those sites that regulate binding and efficacy of alcohols. These binding sites may provide a common motif for binding of alcohols within other classes of ion channels. We will build computational models of binding sites and design specific site-directed mutations to test this hypothesis. These mutations will be expressed and tested by our collaborators, Drs. R. Adron Harris and S. John Mihic, in separately funded experiments. Specifically: Aim 1. We will define specific amino acid residues that determine the "cutoff" length of long-chain alcohols. We have previously shown that mutation of S267 in transmembrane segment 2 (TM2) and A288 in TM3 of the glycine alphal receptor can change the alcohol "cutoff' from heptanol to dodecanol. We will develop computational molecular models that allow us to suggest mutations that will determine additional residues in TM1 and TM4 that may also form "walls" of the putative binding cavities. We will refine our models by iterations in which we optimize the structure of an initial model, use it to predict mutations, test if the model is consistent with the resulting experimental data, and then modify the model in a way that would better fit the data. Aim 2. We will determine the structural requirements of alcohols for potentiation of agonist potency by providing models in which a series of alcohol analogs are covalently linked to site-directed cysteine mutations in the putative binding cavities. Since we will know that a single alcohol analog is bound to the putative site, we can distinguish binding from efficacy. Aim 3. We will define the proximity of amino acid residues important for alcohol potentiation of agonists by building models that predict double site-directed cysteine mutations that are appropriate for cross-linking. We will predict pairs of residues that could be linked by direct disulfide formation or with bi-functional methanethiosulfonate reagents with 1-5 carbon spacers. In summary, these computational studies will provide new knowledge about determinants of alcohol binding and efficacy.

描述(申请人提供)：酗酒和酗酒是主要的健康问题。这些问题的解决很可能需要了解酒精对特定离子通道的影响，以及调节它们的激酶。最近在甘氨酸、GABA、烟碱型乙酰胆碱和5-HT3受体超家族的跨膜结构域中描述了醇的特定结合部位。我们的假设是，酒精结合在由这些受体的跨膜片段所包围的空腔内。我们的目标是定义那些调节酒精结合和功效的位点的属性。这些结合位点可能为醇在其他类型的离子通道中的结合提供了一个共同的基序。我们将建立结合位点的计算模型，并设计特定的定点突变来验证这一假设。这些突变将由我们的合作者R·阿德龙·哈里斯博士和S·约翰·米希克博士在单独资助的实验中表达和测试。具体地说：目标1。我们将定义特定的氨基酸残基，以确定长链醇的“截止”长度。我们先前已经证明，甘氨酸α受体跨膜片段2(TM2)的S267和TM3的A288突变可以将酒精的“截止”从庚醇改变为十二醇。我们将开发计算分子模型，使我们能够建议突变，这些突变将确定TM1和TM4中的额外残基，这些残基也可能形成假定的结合腔的“墙”。我们将通过迭代来改进我们的模型，在迭代中，我们优化初始模型的结构，使用它来预测突变，测试模型是否与最终的实验数据一致，然后以更适合数据的方式修改模型。目的2.我们将通过提供一系列酒精类似物共价连接到假定结合腔中的定点定向半胱氨酸突变的模型来确定酒精对增强激动剂效力的结构要求。因为我们知道一个单一的酒精类似物与假定的位点结合，所以我们可以区分结合和有效性。目的3.我们将通过建立预测适合于交联的双点定向半胱氨酸突变的模型来确定对激动剂的酒精增强重要的氨基酸残基的接近程度。我们将预测可能通过直接二硫键形成或与具有1-5个碳间隔的双功能甲硫磺酸盐试剂连接的残基对。总而言之，这些计算研究将提供关于酒精结合和疗效决定因素的新知识。